Cyclic peptide and a medicament, external preparation and cosmetic comprising said cyclic peptide

ABSTRACT

The present invention is aimed for providing a novel peptide with a high drug efficacy and strong effect, a medicament or external preparation comprising it, specifically a prophylactic or therapeutic for dermatitis, rhinitis or alopecia, or a hair growth stimulant, a hair growing agent, an antipruritic or a skin-care product. The present invention achieved said aim by providing a cyclic peptide having an amino acid sequence expressed by the Formula I or a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence does not have a peptide bond that is not between the amino acids constituting the amino acid sequence.

RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.15/122,753, which is a national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/JP2016/065839, filed May 27, 2016, theentire contents of each of which are incorporated herein by reference intheir entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 22, 2020, isnamed I054170002US01-SUBSEQ-JRV and is 31 kilobytes in size.

TECHNICAL FIELD

The present invention relates to a cyclic peptide and a medicament,external preparation and cosmetic comprising said cyclic peptide.

BACKGROUND ARTS

BNP (brain natriuretic peptide) is a hormone which is synthesized andsecreted in heart (predominantly in ventricles). BNP was isolated fromand identified in pig brain in 1988, and it has been known since thatBNP is secreted from ventricular myocardium of human, etc. An increasein cardiac stress or development of myocardial hypertrophy induces BNPsecretion and increased in blood concentration. In general, BNP hasactivities such as diuretic activity, vasodilation, renin-aldosteronesecretion suppressing action, sympatholytic activity, hypertrophysuppressing action. BNP is considered to a hormone which acts to protectthe myocardium against damages caused by cardiac stress.

Amino acids constituting BNP differ slightly from species to species whoproduce BNP. Nevertheless it been revealed that its structure possessesa cyclic part and a tail part as a common structure (Non-PatentLiteratures 1-2). For instance, wild-type human BNP has been known toconsist 32 amino acid residues, and its fragments and derivatives arefurther proposed (Patent Literatures 1-6).

In Japan, BNP is currently not used as a therapeutic, but widely used inclinical practice as a biochemical marker for cardiac failure. In UnitedStates, however, it is used as a drug for alleviating symptoms ofcardiac failure (trade name: Natrecor®). Recently, it has been proposedto utilize an external preparation comprising BNP for treatingdermatitis, rhinitis, alopecia, etc., and also as a skin-improving agent(Patent Literature7-9).

PRIOR ART REFERENCES Patent Literature

-   [Patent Literature1] JP A 2007-525213-   [Patent Literature2] JP A 2008-509746-   [Patent Literature3] WO 2008/032450-   [Patent Literature4] U.S. Pat. No. 6,028,055-   [Patent Literature5] U.S. Pat. No. 5,114,923-   [Patent Literature6] U.S. Pat. No. 6,818,619-   [Patent Literature7] WO 2011/010732-   [Patent Literature8] WO 2011/024973-   [Patent Literature9] WO 2012/099258

Non-Patent Literature

-   [Non-Patent Literature] N. Akizukia, K. Kangawaa, N. Minaminob, H.    Matsou, FEBS Letters 1991 280 (2)357-362.-   [Non-Patent Literature2] Takei Y, Fukuzawa A, Itahara Y, Watanabe T    X, Yoshizawa Kumagaye K, Nakajima K, Yasuda A, Smith M P, Duff D W,    Olson K R. FEBS Lett. 1997 Sep. 8; 414 (2)377-80.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

As described above, BNP is considered to be a useful substance for thetherapy of various diseases. However, in order to obtain a bettertherapeutic effect there is a need for developing a substance with ahigher drug efficacy and stronger effect and a faster- and longer-actingsubstance.

An improvement in the duration and fast-acting properties of the drugefficacy and effect is quite meaningful in light of reducing requirednumber/frequency of treatment, relieving the sufferings by thepatient/user, and improving the quality of life (QOL) and the mental,physical, social or intellectual satisfaction in dairy living of theuser. For example, dermatitis is often accompanied by symptoms such asan itch, infiltration or hot flush in the affected site; the sufferingsby the patient will be reduced if these symptoms can quickly bealleviated. When an alopecia therapeutic, hair growing agent or hairgrowth stimulant is employed, the QOL of the user can be improved if thetherapeutic effect, hair growth-stimulating or hair growing effect canbe achieved immediately.

The inventors focused on these points and tried to search for a novelsubstance by reference to the structure of BNP.

Accordingly, the object of the present invention is to provide a novelpeptide having a strong drug efficacy and effect, and a medicament or anexternal preparation comprising such peptide, specifically aprophylactic or therapeutic for dermatitis, rhinitis and alopecia, and ahair growth stimulant, a hair growing agent, an antipruritic, a cosmeticand a skin-care product, etc.

Means to Solve the Problems

It was previously considered that the tail part of the wild-type BNPpeptide structure (which is composed of a cyclic part and tail part)plays at least certain important role in the binding and selectivity ofBNP to its receptor. However, the inventors focused on the cyclic partof said structure, deleted the tail part, and made an intensive researchon the resulting cyclic peptide. Accordingly we have obtained totallynew findings including the effects of such cyclic peptide, its high drugefficacy, faster-acting and longer-lasting effect, and further continuedthe study and finally completed the invention.

Accordingly, the present invention relates to the followings:

[1] A cyclic peptide having an amino acid sequence expressed by FormulaI:

wherein,X¹ denotes Gly, Val, Ala, Ser or Thr,X² denotes Arg, Gln or His,X³ denotes Lys or Arg,X⁴ denotes Met, Leu or Ile,X⁵ denotes Ile or Val,X⁶ denotes Ser or Gly,X⁷ denotes Ser or Ala,X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met,X⁹ denotes Ser, Val, Ala or Thr,X¹⁰ denotes Gly or Arg,X¹¹ denotes Leu, Met, Ile, Val or Ala,X¹² denotes Gly, Ser or Ala, andthe line connecting two Cys denotes a disulfide bond,and wherein the amino acid sequence does not have a peptide bond that isnot between the amino acids constituting the amino acid sequence,or a derivative thereof or a pharmaceutically acceptable salt thereof.[2] The cyclic peptide according to [1], wherein

X² denotes Arg,

X⁴ denotes Met,

X⁶ denotes Ser,

X⁷ denotes Ser, and

X¹⁰ denotes Gly,

or a derivative thereof or a pharmaceutically acceptable salt thereof.[3] The cyclic peptide according to [1], wherein the amino acid sequenceis selected from the amino acid sequences expressed by Formula(I-a)-Formula (I-e):

wherein, the line connecting two Cys denotes a disulfide bond,or a derivative thereof or a pharmaceutically acceptable salt thereof.[4] The cyclic peptide according to any one of [1]-[3] or a derivativethereof or a pharmaceutically acceptable salt thereof, wherein thederivative is substituted by a substituent which is capable of replacinga hydrogen atom, hydroxyl group, carboxy group, amino group or iminogroup in the cyclic peptide.[5] The cyclic peptide or a derivative thereof or a pharmaceuticallyacceptable salt thereof, which is formed by deleting 1 to 4 amino acidsin the cyclic peptide expressed by any one of the Formulae (I-a)-(I-e)according to [3], or by replacing them with or adding them other aminoacids, and which has an equal function with the cyclic peptide expressedby each of said formulae.[6] An external preparation comprising one or more cyclic peptidesaccording to any one of [1]-[5] and/or a derivative thereof and/or apharmaceutically acceptable salt thereof.[7] The external preparation according to [6], wherein the externalpreparation is an ingredient for a dermatitis therapeutic, dermatitisprophylactic, antipruritic, antiphlogistic, epidermis regenerationaccelerating agent, wound epithelialization-accelerating agent orskin-care product.[8] The external preparation according to [7], wherein the skin-careproduct is for moisturizing, and/or for preventing or improving roughskin, and/or for sebum/acne care, and/or for irritationalleviation/anti-inflammation, and/or for skin-lightening, and/or foranti-aging, and/or for preventing/alleviating ultraviolet lesion, and/orfor slimming, and/or for skin-cleansing.[9] The external preparation according to [6], wherein the externalpreparation is a bath agent, a body-cleansing agent or a hair-cleansingagent.[10] The external preparation according to [6], wherein the externalpreparation is an alopecia therapeutic, an alopecia prophylactic, a hairgrowing agent and/or a hair growth stimulant.[11] The external preparation according to [6], wherein the externalpreparation is a rhinitis therapeutic and/or a rhinitis prophylactic.[12] The external preparation according to [6], wherein the externalpreparation is a cosmetic.[13] The external preparation according to any one of [6]4121, whereinthe formulation is a solid, semi-solid, powder, liquid, spray, ointment,cream, emulsion, gel or patch formulation.[14] The external preparation according to any one of [6]4131, whereinthe external preparation is used as a pharmaceutical product, aquasi-drug or a cosmetic product.[15] A use of the cyclic peptide according to any one of [1]-[4] and/ora derivative thereof and/or a pharmaceutically acceptable salt thereoffor preparing the external preparation.[16] A method of using the external preparation comprising applying theexternal preparation according to any one of [6]-[14] to the skin and/ormucosa of a subject.[17] The method of using the external preparation according to [16],wherein the mucosa is labial, oral, nasal, ocular or vaginal mucosa.[18] The method of using the external preparation according to [16],wherein the method is a method of treating and/or preventing dermatitis,a method of alleviating or resolving itch, a method of treatingeczematous or other erosion or ulcer, or a method for skin-care.[19] The method of using the external preparation according to [16],wherein the method is a method of treating and/or preventing alopecia,and/or a method of stimulating hair growth, and/or a method of growinghair.[20] The method of using the external preparation according to [16],wherein the method is a method of treating and/or preventing rhinitis.[21] A medicament comprising one or more cyclic peptides according toany one of [1]-[5] or a derivative thereof or a pharmaceuticallyacceptable salt thereof.[22] The medicament according to [21], wherein the medicament is atherapeutic for hypertension, unstable angina, acute myocardialinfarction, edematous diseases, renal failure, cardiac failure, immunediseases, obesity or metabolic syndrome.

The cyclic peptide described herein (hereinafter also referred to as“BNP cyclic peptide”, “B ring” or “B ring-compound”) is, as describedabove, derived from wild-type BNP. The wild-type BNP encompasses BNPfrom human, as well as BNPs from monkeys, pigs, birds and rats.Therefore, the B ring-compound also encompasses the B ring-compound fromhuman, as well as those from monkeys, pigs, birds and rats.

Effect by the Invention

According to the present invention, novel cyclic peptides andcompositions comprising the same can be provided. Such compositions areapplied to an external preparation for preventing or treatingdermatitis, rhinitis or alopecia, and further applied to a hair growthstimulant, a hair growing agent or an antipruritic, each of which areprovided as a medicament, a quasi-drug, a skin-care product, or acosmetic product. When an “external preparation” is referred alone inthe present invention, it means an agent that is applied to skin ormucosa, whose utility is not limited to a medicament, quasi-drug,skin-care product and cosmetic product.

In general, the external preparation of the present invention has asignificantly higher efficacy against dermatitis as compared to aconventional steroid external preparation, as well as an excellentimmediate effect such that the symptoms start to be improved withinthree minutes in general. Its effect is great and long-lasting, and theamelioration period is long.

Furthermore, the external preparation of the present invention can, uponbeing applied onto the subject's skin or mucosa suffering a dermatitis,quickly relieve or eliminate perceptible symptoms that are or can becaused by dermatitis such as pruritus, soreness (pain), hot sensation,tautness, infiltration and erythema, improving the symptoms of thedermatitis.

Furthermore, the external preparation of the present invention exerts amoisturizing effect on the applied site, while exerting an effect toimprove skin texture where the corneum layer is present at the appliedsite. It can thus exerts effects to improve skin texture, improves dryor rough skin, softens and moisturizes skin, reduce and diminishwrinkles, and improve roughened lip.

The external preparation of the present invention also has an effect ofstimulating hair growth or promoting hair growth and at the same timepreventing hair loss at the applied site when being applied to adecalvant site or a site where hair growth is stimulated. In this case,stimulated hairs tend to become terminal hairs and not white hairs.These effects are exhibited relatively quick as compared to other activeagents that have previously been used in the therapeutics for alopecia,e.g., BNP, and the obtained effects are more significant.

Yet the external preparation of the present invention has an immediate,large and long-lasting effect effect on rhinitis with a longamelioration period.

Besides, since the cyclic peptide of the invention is a peptide sharinga part of its structure with BNP which is a hormone inherent in body, itcauses little concern about side effects. It is also considered to havea small influence on homodynamics as long as used in an appropriateamount or used externally onto skin, etc. It therefore can beadministered for prolonged period to a patient in need thereof, e.g., apatient with chronic dermatitis. Moreover, the external preparation ofthe invention causes no irritation when being taken externally and canbe applied safely to a patient with sensitive skin, as well as to achild or woman, on face or neck, etc.

The reason why the cyclic peptide of the invention has a superior drugefficacy and effect as compared with wild-type BNP is not yet clear.However, according to our in silico analysis, it is suggested that thecyclic peptide of the invention is more easily and strongly bound toNPR-A receptor (GC-A) as compared with wild-type BNP. It is thuspredicted that the cyclic peptide would have an excellent drug efficacyand effect, an excellent immediate effect in particular. As describedabove, B ring-compound is derived not only from human, but there alsoare B rings from animals of other species such as monkeys, pigs, birdsand rat, and it has been confirmed that they have similar structures.From the results of the structural analysis described hereinbelow, it isstrongly speculated that all these B rings exert similar effects asthose of the B ring-compound from human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

A diagram showing the result of applying BNP cyclic peptide (A) or a gelformulation (B) on either left or right side of the face of a subjecthaving large wrinkles on the face.

FIG. 2

A diagram showing the effect before and after the application of eitherB ring gel formulation or BNP gel formulation to a patient with femalepattern alopecia and alopecia pityroides.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is explained in detail based on itssuitable embodiments.

1. The cyclic peptide, a derivative thereof and a pharmaceuticallyacceptable salt thereof Firstly, the cyclic peptide of the invention, aderivative thereof and a pharmaceutically acceptable salt thereof areexplained.

The cyclic peptide of the present invention has an amino acid sequenceexpressed by Formula I:

wherein,X¹ denotes Gly, Val, Ala, Ser or Thr,X² denotes Arg, Gln or His,X³ denotes Lys or Arg,X⁴ denotes Met, Leu or Ile,X⁵ denotes Ile or Val,X⁶ denotes Ser or Gly,X⁷ denotes Ser or Ala,X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met,X⁹ denotes Ser, Val, Ala or Thr,X¹⁰ denotes Gly or Arg,X¹¹ denotes Leu, Met, Ile, Val or Ala,X¹² denotes Gly, Ser or Ala,the line connecting two Cys denotes a disulfide bond,and wherein the amino acid sequence does not have a peptide bond that isnot between the amino acids constituting the amino acid sequence.

Such cyclic peptide is, similar to previously known BNPs, considered tobind to a receptor NPR-A (also known as GC-A) having a guanylate cyclasedomain and promote the production of cyclic guanosine monophosphate(cGMP), and has activities such as, for example, diuretic action,vasodilation, renin-aldosterone secretion suppressing action,sympatholytic activity and hypertrophy suppressing action. It has asuperior drug efficacy and effect, particularly an excellent immediateeffect, as compared to BNP.

In addition, as described hereinbelow, the above cyclic peptide can beused as an ingredient of an external preparation for a dermatitistherapeutic/prophylactic, a rhinitis therapeutic/prophylactic, analopecia therapeutic/prophylactic, a hair growing agent, a hair growthstimulant, an antipruritic, etc., or as an ingredient of a skin-careproduct, a quasi-drug or a cosmetic product. The above cyclic peptidecan also be used as an alternative for BNP in an medicament utilizingthe activity of BNP as above, e.g., in an medicament for hypertension,unstable angina, acute myocardial infarction, edematous diseases, renalfailure, cardiac failure, immune diseases, obesity or metabolicsyndrome.

In another embodiment of the present invention, in the cyclic peptide ofthe invention, X¹-X¹² in the Formula (I) may be defined as one or moreselected from the group consisting of following (1)-(12):

(1) X¹ denotes Gly, Val, Ala, Ser or Thr,(2) X² denotes Arg, Gln or His,(3) X³ denotes Lys or Arg,(4) X⁴ denotes Met, Leu or Ile,(5) X⁵ denotes Ile or Val,(6) X⁶ denotes Ser or Gly,(7) X⁷ denotes Ser or Ala,(8) X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met,(9) X⁹ denotes Ser, Val, Ala or Thr,(10) X¹⁰ denotes Gly or Arg,(11) X¹¹ denotes Leu, Met, Ile, Val or Ala, and(12) X¹² denotes Gly, Ser or Ala.

A preferred cyclic peptide of the present invention is a cyclic peptideof Formula (I), wherein X² denotes Arg, X⁴ denotes Met, X⁶ denotes Ser,X⁷ denotes Ser, and/or X¹⁰ denotes Gly (SEQ ID NO: 2).

Furthermore, in another embodiment of the invention, in the cyclicpeptide of the invention, the amino acid sequence expressed by Formula(I) may be selected from SEQ ID NOs: 3-8 and SEQ ID NOs: 16-75.

Furthermore, in the cyclic peptide of the invention, the amino acidsequence expressed by Formula (I) is preferably selected from the aminoacid sequences expressed by the Formula (I-a)-Formula (I-e):

wherein, the line connecting two Cys denotes a disulfide bond.

Each of the amino acid sequences expressed by these Formulae (I-a)-(I-e)is the cyclic part of human BNP (Formula (I-a)), swine BNP (Formula(I-b)), rat BNP (Formula (I-c)), rabbit BNP (Formula (I-d)) and murineBNP (Formula (I-e)), respectively. Therefore, the cyclic peptide havingsuch an amino acid sequence will exhibit the aforementioned effects withmore certainty. Because swine BNP cyclic peptide is consistent withthose of avian (SEQ ID NO: 8,Cys-Phe-Gly-Arg-Arg-Ile-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Met-Gly-Cys,wherein, 1st Cys and 17th Cys form a disulfide bond), bovine (SEQ ID NO:9, Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys,wherein, 1st Cys and 17th Cys form a disulfide bond), feline (SEQ ID NO:10, Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys,wherein, 1st Cys and 17th Cys form a disulfide bond), canine (SEQ ID NO:11, Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys,wherein, 1st Cys and 17th Cys form a disulfide bond), and ovine (SEQ IDNO: 12,Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys,wherein, 1st Cys and 17th Cys form a disulfide bond), BNP cyclicpeptides derived from these organisms also have similar effects as thecyclic peptide of the present invention. Among those mentioned above, itis particularly preferred that the amino acid sequence expressed byFormula (I) is the amino acid sequence expressed by Formula (I-a). Inanother embodiment of the invention, the cyclic peptide may be expressedby SEQ ID NOs: 16-75.

The cyclic peptide of the present invention encompasses a derivatizedform of said cyclic peptide. Such a derivative can be used as such, asan active substance, or also used as a prodrug.

A derivative of the present invention can be obtained by adding(modifying) a known substituent to certain group of an amino acid in thecyclic peptide such as, for example, a hydrogen atom, hydroxyl group,carboxy group, amino group or imino group, or replacing it with a knownreplaceable substituent. Modification include, but not limited to,chemical modifications such as, for example, glycosylation, acetylation,phosphorylation and lipidation, to an amino acid within the cyclicpeptide.

The addition (modification) or replacement of an amino acid within thecyclic peptide can occur in one group, or can occur in more than onegroup at the same time. Any known substituents can be used as long asbeing capable of replacing above groups and it goes without saying thatsuch substituents naturally include, for example, protecting groups suchas BOC.

The cyclic peptide of the invention further includes a mutated form ofsaid cyclic peptide. Namely, a mutant of the present invention can beobtained by deleting an amino acid in the cyclic peptide, replacing itwith or adding it another amino acid. The number of amino acid to bedeleted, replaced with other amino acids or added is 4 or less, morepreferably 3 or less, even more preferably 2 or less, and particularlypreferable 1 or less. It also goes without saying that the deletion,replacement or addition of amino acids in the cyclic peptide may occurconcurrently and independently to each other.

An amino acid that is capable of being replaced with another amino acidcan be, in the case of human BNP (Formula (I-a)), exemplified asfollows, without being limited thereto. The third amino acid from left,Gly, may be replaced with either Val, Ala, Ser or Thr. The forth aminoacid from left, Arg, may be replaced with either Gln or His. The fifthamino acid from left, Lys, may be replaced with Arg. The sixth aminoacid from left, amino acid, Met, may be replaced with either Leu or Ile.The ninth amino acid from left, Ile, may be replaced with Val. Thetwelfth amino acid from left, Ser, may be replaced with either Gln, Val,Ala, Thr, Leu, Ile or Met. The thirteenth amino acid from left, Ser, maybe replaced with either Val, Ala or Thr. The fourteenth amino acid fromleft, Gly, may be replaced with Arg. The fifteenth amino acid from left,Leu, may be replaced with either Met, Ile, Val or Ala. The sixteenthamino acid from left, Gly, may be replaced with either Ser or Ala. Table32 summarizes examples of replaceable amino acids in the cyclic peptideof the invention, though the invention will not be limited thereto.

Cyclic peptides in which one amino acid has been replaced include suchas, for example, SEQ ID NOs: 16-44, though the invention will not belimited thereto. Cyclic peptides in which two amino acids have beenreplaced include such as, for example, SEQ ID NO: 45-58, though theinvention will not be limited thereto. Yet cyclic peptides in whichthree amino acids have been replaced include such as, for example, SEQID NOs: 59-70, though the invention will not be limited thereto.Furthermore, cyclic peptides in which four amino acids have beenreplaced include such as, for example, SEQ ID NOs: 71-75, though theinvention will not be limited thereto. It also goes without saying thatfive or more amino acids can be replaced by appropriately combining theaforementioned amino acid.

Deletion of one to several amino acids can take place in similar mannerto the replacement of the aforementioned amino acids with other aminoacids.

The number of amino acid which are to be deleted or replaced with otheramino acids or added may be determined such that the cyclic peptide willhave 80% homology, preferably 90% homology to the cyclic peptide of theinvention.

In addition, the present invention may be a mutant as described above,and a derivative thereof. As long as it retains the effect of theinvention, any mutant or a derivative thereof is encompassed in thecyclic peptide according to the invention. In another embodiment, it hasat least a BNP activity. For purpose of improving the activity of thecyclic peptide of the invention, prolonging the effect of the invention,and/or increasing storage stability of the cyclic peptide of theinvention, the cyclic peptide of the invention or the amino acidsconstituting said peptide may be altered in an appropriate manner. Forexample, an amino acid in the cyclic peptide of the invention may bechemically modified, some amino acids constituting the cyclic peptidemay be deleted or replaced with other amino acids, and/or new aminoacids may be added.

For example, the C-terminal group —COOH of one Cys of the cyclic peptidemay be replaced with —COOR¹, —CONHR¹ or —CONR¹², and/or the N-terminalgroup NH₂ of the other Cys of the cyclic peptide may be replaced with—NHC(O)R¹ or —N(C(O)R¹)₂. Here, each appearance of R¹ is independently abranched or straight hydrocarbon group or an alkylene glycol chain orsugar chain having 1 to 20 carbon atoms. The number of carbon atoms inR¹ is preferably 1 to 10, more preferably 1 to 5, yet more preferably 1to 2.

Pharmaceutically acceptable salts include, without being particularlylimited, as the cyclic peptide of the invention or a derivative, such asfor example, a salt with an inorganic base, a salt formed with anorganic base, a salt formed with an inorganic acid, a salt formed withan organic acid, a salt formed with a basic or acidic amino acid.Examples of suitable salt formed with inorganic base include such as,for example, an alkaline metal salt such as a sodium salt and potassiumsalt; an alkaline earth meal salt such as a calcium salt and magnesiumsalt; and an aluminum salt and ammonium salt. Examples of suitable saltformed with organic base include such as, for example, a salt with analkyl amine such as trimethyl amine or triethyl amine; a salt formedwith a heterocyclic amine such as pyridine and picoline; a salt formedwith an alkanol amine such as ethanol amine, diethanol amine andtriethanol amine; a salt formed with a cycloalkyl amine such ascyclohexyl amine and dicyclohexyl amine; a salt formed with an alkylenediamine derivative such as N,N′-dibenzylethylenediamine. Examples ofsuitable salt formed with inorganic acid include such as, for example, asalt formed with hydrochloric acid, hydrobromic acid, nitric acid,sulfuric acid or phosphoric acid. Examples of suitable salt formed withorganic acid include such as, for example, a salt formed with amonocarboxylic acid such as formic acid, acetic acid, trifluoroaceticacid and propionic acid; a salt formed with a polyvalent carboxylic acidsuch as fumaric acid, oxalic acid and maleic acid; a salt formed with aoxycarboxylic acid such as tartaric acid, citric acid, succinic acid andmalic acid; a salt formed with a sulfonic acid such as methane sulfonicacid, benzene sulfonic acid and p-toluene sulfonic acid; and a saltformed with benzoic acid. Examples of suitable salt formed with aneutral amino acid include such as, for example, a salt formed withglycine, valine or leucine; examples of suitable salt formed with abasic amino acid include such as, for example, a salt formed witharginine, lysine or ornithine; and examples of suitable salt formed withan acidic amino acid include such as, for example, a salt formed withaspartic acid acid or glutamic acid.

Among those mentioned above, a cyclic peptide composed of the amino acidsequence expressed by Formula (I) or a pharmaceutically acceptable saltis preferred. Namely, it is preferred that the amino acid sequenceexpressed by Formula (I) is not replaced.

A method for producing the cyclic peptide of the present invention, aderivative and pharmaceutically acceptable salt thereof may employ,without being particularly limited, for example, any known chemicalsynthetic or genetic engineering methods.

When the amino acid sequence as above is chemically synthesized, it maybe synthesized by any chemical synthetic method, or any known method forpeptide synthesis, for example, or solid-phase or liquid-phase syntheticmethod. Moreover, any commercial synthesizer (e.g., SHIMADZUCorporation: PSSM-8) may be used for synthesis.

A disulfide bond can be formed in the amino acid sequence, for example,by DMSO oxidation method or iodine oxidation method without beingparticularly limited. In this case, an intramolecular disulfide bond canbe formed by treating a free sulfhydryl group or a sulfhydryl groupprotected by a protecting group with either DMSO or iodine (I2) toresult in said cyclic peptide.

A protecting group includes such as, for example, 4-methylbenzyl group(Bzl (4Me)), trityl group (Trt), tert-butyl group, N-(acetyl)aminomethyl group (Acm). Deprotection can be carried out throughappropriate treatment corresponding to these protecting group, forexample, for 4-methylbenzyl group by treating with a strong acid, andfor N-(acetyl) aminomethyl group by treating with iodine.

Next, if necessary, the cyclic peptide is derivatized to result in aderivative. Derivatization can be carried out by known method.Alternatively, a derivative of the cyclic peptide can be produced at thetime of peptide synthesis by introducing in advance a substituent intothe amino acid constituting the cyclic peptide.

Then, if necessary, a pharmaceutically acceptable salt is produced byion exchange of the cyclic peptide or derivative thereof. Ion exchangecan be carried out, for example, by bringing into the contact the cyclicpeptide or derivative thereof with a desired acid or base.

On the other hand, when the peptide is synthesized by geneticengineering, it can be synthesized by any known method such as, forexample, methods described in Sambrook J. et al., Molecular Cloning, ALaboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press(2012)). For instance, a DNA fragment coding for the amino acid sequenceexpressed by Formula (I) is prepared at first. Preparation of the DNAfragment can be performed, for example, if it is a case in which the DNAfragment codes for the amino acid sequence expressed by Formula (I-a),by amplifying the DNA fragment by PCR using a vector comprising afull-length human BNP gene as template and primers designed tosynthesize a defined DNA region. Alternatively, a DNA fragment can bechemically synthesized.

Then, the amplified DNA fragment is ligated into an appropriate vectorto obtain a recombinant vector for protein expression. Next, the vectorfor protein expression is allowed to be taken up by target cells and thetransformed cells are selected. Finally, the protein produced by thecells (i.e., the protein composed of the amino acid sequence expressedby Formula (I)) is collected.

Formation of disulfide bond, derivatization and salt formation in thecollected protein can be carried out as described above.

The identification of the compound of interest such as a cyclic peptidecan be confirmed by known procedures such as, for example, reverse-phaseHPLC or mass spectroscopy.

The presence or absence of BNP activity in the obtained compound canreadily be confirmed by known means. For instance, it can be confirmedby examining cGMP producing activity in NPR-A receptor-expressing cells.

2. External Preparation

Secondly, the external preparation of the present invention isexplained.

The external preparation of the present invention comprises one or morecyclic peptide and/or a derivative thereof and/or a pharmaceuticallyacceptable salt thereof as described above. If two or more cyclicpeptides are used, the number of different cyclic peptide to be mixed isnot limited, though 2 to 3 are preferred in terms of preparation costand convenience. It includes SEQ ID NO: 19, 29, 33, 36, 39 and 43, forexample, but the invention is not limited to these and these cyclicpeptide can appropriately be combined as long as it does not interferethe effect of the invention.

2.1 Application

The external preparation of the present invention can be used forfollowing uses, for example, without being particularly limited, and ineach case exerts remarkable effect which had not previously beenachieved. The external preparation of the present invention is notlimited to any particular use, and can be used as a pharmaceuticalproduct, a quasi-drug and/or a cosmetic product depending on its drugefficacy and effect.

Hereinbelow, the invention will be described in detail for each of itsuse.

(1) Therapeutic/Prophylactic for Dermatitis

The external preparation of the present invention can be a a therapeuticand/or prophylactic for dermatitis.

The external preparation of the present invention can, upon beingapplied onto the subject's skin or mucosa suffering dermatitis, quicklyrelieve or eliminate various perceptible symptoms and conditions thatare or can be caused by dermatitis such as pruritus, soreness (pain),hot sensation, tautness, erythema, infiltration, papule,lichenification, crust, exudation or skin desiccation, improving thesymptoms of dermatitis. In addition, the external preparation of thepresent invention does not cause any irritation at the site ofapplication.

Furthermore, such action of the external preparation comprising thecyclic peptide of the invention is effected more quickly and forprolonged time period as compared to that of an external preparationcomprising BNP having the tail part. Although reasons for theseadvantageous effects of the cyclic peptide over such BNP are not clear,the cyclic peptide of the invention is considered to have anadvantageous structure for binding to its receptor (NPR-A receptor) overBNP, thereby enabling faster binding to the NPR-A receptor in thevicinity of the affected site and at the same time prolonging itsbinding time. Moreover, it is considered that its relatively quickbinding to the NPR-A receptor near the affected site can prevents thecyclic peptide from diffusing out of the affected site via bloodstream,etc., enabling it to stay around the affected site for relatively longtime.

The external preparation of the present invention does not only suppressor prevent inflammation in dermatitis but also acts to restore and/orretain the barrier function of skin. Here, skin's barrier function worksfor protecting skin against entry of stimuli and saprophytes fromexternal environment or for retaining moisture. The external preparationrestores the barrier function, and thus able to prevent the progress orexacerbation of inflammation. The barrier function of skin is greatlyaffected by the alignment state of corneocytes in the corneum, i.e.,state of skin texture and moisturization. The external preparation ofthe present invention acts to improve skin texture and to moisturizeskin. Therefore, its effects of restoring and maintaining skin barrierfunction are also obvious.

The external preparation of the present invention is also effective onskin symptoms commonly called acne, i.e., such as comedones, red papule,pustula, cysts/tuberosity.

Moreover, a quick relief or elimination of perceptible symptoms orconditions will reduce the burden of the subject (patient) and improvethe QOL of the patient, while preventing the patient from being botheredby itch or soreness and from acting to damage the affected site, forexample, touching or scratching. This effect of the external preparationof the present invention to relieve or eliminate perceptible symptoms orconditions is, in general, exhibited within 10 minutes, preferablywithin 5 minutes, more preferably within 3 minutes after application.

In addition, the external preparation of the present invention can treatdermatitis in which steroid dermatosis has been developed, or dermatitisthat is intractable by other common drugs for dermatitis therapy such assteroid and tacrolimus, for example dermatitis for which a sufficienttherapeutic effect cannot be achieved by these formulations, ordermatitis which is resistant to these formulations, or dermatitis forwhich the use of these formulations is not suitable or desirable.Conventional widely-used steroid external preparations have significantproblems upon terminating application that the severity returns to thepre-application level and that a rebound phenomenon may occur andexacerbate the condition. The external preparation of the presentinvention has no such problems as a rebound phenomenon.

Dermatitis is in general a disease which causes inflammation in skin ormucosa. Dermatitis is normally accompanied with one or more symptomsselected from acute eczema symptoms such as erythema, infiltrativeerythema, papule, vesicle, pustula, infiltration, incrustation anddesquamation; chronic eczema symptoms such as lichenification andpigmentation; and scales, crust (scab) attachment, scratching, scratchscar, prurigo nodularis, erosion, edema, oozing and squamatization.

Dermatitis herein is not particularly limited as long as it is a diseasewhich is accompanied with inflammation in skin or mucosa and includessuch as, for example, eczema and atopic dermatitis such as chroniceczema, dyshidrotic eczema, infantile xerotic eczema; contact dermatitissuch as allergic contact dermatitis and primary irritant contactdermatitis; seborrheic dermatitis; asteatotic dermatitis;autosensitization dermatitis; stasis dermatitis; urticaria such asallergic urticaria (e.g., alimentary urticaria and drug-inducedurticaria) and nonallergic urticaria (e.g., physical urticaria, solarurticaria and cholinergic urticaria); insect bite; drug eruption;psoriasis such as plaque psoriasis, guttate psoriasis, erythrodermicpsoriasis, pustular psoriasis and psoriasis arthropathica; prurigo suchas chronic prurigo, acute prurigo, gestational prurigo and nodularprurigo; rosacea; rosacea-like dermatitis; cutaneous vasculitis such ascutaneous allergic vasculitis; cutaneous pruritus such as systemiccutaneous pruritus, localized cutaneous pruritus, senile cutaneouspruritus and gestational pruritus; solar dermatitis; erythrosis;nummular dermatitis; localized scratching dermatitis; perioraldermatitis; pompholyx; keratosis pilaris; lichen planus, dyshidroticeczema, dyshidrosis, miliaria and acne vulgarisacne vulgaris. Theexternal preparation of the present invention can be applied to thetherapy and prophylaxis of any of these diseases.

The external preparation of the present invention exerts an excellenteffect especially on eczema, atopic dermatitis, contact dermatitis,seborrheic dermatitis, insect bite, allergic or nonallergic urticariaand psoriasis, preferably on eczema, atopic dermatitis, contactdermatitis and insect bite, more preferably on eczema and atopicdermatitis. Therefore, the external preparation of the present inventioncan be used for the purpose of the therapy and prophylaxis of at leastone or more dermatitis selected from above-mentioned group.

(2) Ingredients of Cosmetic or Skin-Care Product

The external preparation of the present invention may be an ingredientfor a cosmetic or skin-care product.

The external preparation of the present invention will, upon beingapplied to skin and/or mucosa, provide moisture to the applied site andexert moisturizing effect, preventing skin from dryness and roughness.In cases where corneum is present at the site of application, it exertsan effect of improving skin texture there. It also provides and retainsmoderate elasticity and flexibility of skin and mucosa, softens skin,gives skin and mucosa resilience and firmness. Moreover, it improveswrinkles (including fine and large wrinkles) and flabbiness at theapplied site, and further diminishes the appearance of dullness andspots. By diminishing dullness and spots away, it consequently providesa skin-lightening effect.

As a result, the external preparation of the present invention can beused for the purpose of maintaining or improving skin and/or mucosalcondition, without being particularly limited, for example, forobtaining at least one effects selected from a group consisting ofpreventing or improving dry skin, skin roughness, sensitive skin,roughened lips and appearance of large and fine wrinkles, maintainingskin or mucosal condition, anti-aging, and skin-lightening, or obtainingfor at least one effects selected from the above effects. Skin roughnessherein includes miliaria, chilblain, cracked skin, chapped skin, acne,diaper rashes, festering, chafed inner thighs and razor burns.

Specific utilities of the external preparation of the present inventionas an ingredient for the cosmetic or skin-care product includes, withoutbeing particularly limited, for example, cosmetics for make-up such asface lotion, emulsion, serum, cream, cold cream, gel, mask, pack,powder, hand soap, perfume, deodorant, as well as foundation, facepowder, eye shadow, eyeliner, mascara, eyebrow, blush, makeup base, lipstick, lip cream and nail polish, and furthermore cosmetics for hairsuch as shampoo, rinse, conditioner, hair color, hair tonic, stylingagent and perm chemical, body cleansers such as face wash, cleansing andbody soap, skin-care products such as body powder, after-shave lotionand pre-shave lotion, and bath agents.

The above effects of the external preparation of the present inventionare quickly exhibited, within 10 minutes in general, preferably within 5minutes, more preferably within 3 minutes after application depending onthe type of the effect.

The above effects of the external preparation of the present inventionlast for relatively long time. In general, the effect lasts for 4 hoursor more, preferably, 8 hours or more, more preferably 24 hours or moreafter it started to effect depending on the type of the effect.

(3) Antipruritic

The external preparation of the present invention can be anantipruritic.

As mentioned above, the external preparation of the present invention issuitable for using as antipruritic because it quickly exerts anexcellent antipruritic effect at the applied site when being applied toskin or mucosa.

The antipruritic effect of the external preparation of the presentinvention as mentioned above is exhibited within 10 minutes in general,preferably within 5 minutes, more preferably within 3 minutes afterapplication.

(4) Alopecia Therapeutic, Alopecia Prophylactic, Hair Growing Agent andHair Growth Stimulant

The external preparation of the present invention can also be analopecia therapeutic, alopecia prophylactic, hair growing agent and/orhair growth stimulant.

When the external preparation of the present invention is applied to asite of hair loss or a site where hair growth is to be stimulated, itacts to prevent hair loss and promote hair growth stimulation and hairgrowing at the applied site. It has effects such as nourishing hair,promoting hair growth, and improving or preventing hair thinning at theapplied site. In this case, hairs to be stimulated to grow tend tobecome terminal hairs or hairs which are not white hairs. These effectsare exhibited in relatively early stage as compared to other activeingredients previously used in alopecia therapeutics e.g., BNP, and theeffects obtained are significant.

The external preparation of the present invention also has an improvingor preventing effect on dermatitis as mentioned above. Therefore, it canimprove or prevent skin inflammation associated with alopecia. Sucheffects are particularly advantageous when alopecia has been exacerbateddue to skin condition of the site of application (such as scalp).

Moreover, the external preparation of the present invention exertsmoisturizing and skin texture-improving effects at the applied site whenbeing applied to skin as mentioned above. It can remove and suppressdandruff and itching, while giving moisture to hair and scalp, improvingand preventing dryness and keeping hair and scalp healthy. It also canimprove seborrhea.

The external preparation of the present invention also acts to alleviateor eliminate the perceptible symptoms or conditions as mentioned above,thereby reducing the burden of the subject (patient) and improving QOLof the patient. Moreover, it can prevent the patient from being botheredby itch or soreness and acting to damage the affected site, for example,touching or scratching, thereby preventing exacerbation of skincondition which may induce alopecia.

When the external preparation of the present invention is used asalopecia therapeutic or prophylactic, applicable alopecia includes suchas, for example, alopecia as listed below, without being particularlylimited. The external preparation can be used for the purpose oftreating or preventing one ore more of these alopecia.

(Acquired Alopecia)

(i) Alopecia without accompanying scarring or skin lesion (alopeciaareata, male pattern alopecia, seborrheic alopecia, alopecia pityroides,female pattern alopecia, gestational alopecia, malignant alopecia,senile alopecia, alopecia totalis, alopecia areata multilocularis,ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopeciaand radiation exposure-induced alopecia, traumatic/mechanical alopecia,malnutrition/metabolic disorder associated alopecia, endocrinedysfunction associated alopecia and telogen effluvium (post partumalopecia, alopecia after high fever)).(ii) Alopecia observed on skin lesion or pathologic skin(infection-induced alopecia, tumor-induced alopecia,inflammation-induced alopecia)(iii) Scarring alopecia (skin infection-induced alopecia, alopeciainduced by infiltration of inflammatory cells)

(Congenital Alopecia)

Diffuse alopecia, congenital atrichia, alopecia in hereditary syndromes,localized alopecia, phakomatosis, aplasia cutis, congenital alopeciatriangularis.

Among those mentioned above, the external preparation of the presentinvention exerts an excellent effect particularly on acquired alopecia,preferably on alopecia without accompanying scarring or skin lesion,more preferably on alopecia areata, male pattern alopecia, seborrheicalopecia, alopecia pityroides, female pattern alopecia, gestationalalopecia, malignant alopecia, senile alopecia, alopecia totalis,alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancerchemotherapy-induced alopecia and radiation exposure-induced alopecia,traumatic/mechanical alopecia, malnutrition/metabolic disorderassociated alopecia, endocrine dysfunction associated alopecia andtelogen effluvium. Therefore, the external preparation of the presentinvention can be used for the purpose of treating or preventing at leastone or more alopecia selected from the above-mentioned group.

(5) Rhinitis Therapeutic and/or Prophylactic

The external preparation of the present invention can also be a rhinitistherapeutic and/or prophylactic.

Rhinitis is a disease caused by mucosal inflammation in nasal cavityand/or paranasal cavity and induces main symptoms such as nasalobstruction, rhinorrhea, sudden recurrent sneezing, as well as symptomssuch as pruritus. In the present invention, “rhinitis” does not onlyincludes rhinitis in a narrow definition which are accompanied withmucosal inflammation in nasal cavity, but also includes sinusitisaccompanied with mucosal inflammation in paranasal cavity.

The external preparation of the present invention can improve or preventvarious symptoms associated with rhinitis such as nasal obstruction,rhinorrhea, sneezing and pruritus, when being applied to nasal cavitymucosa and/or paranasal cavity mucosa. Especially these effects areexhibited quickly and last for a prolonged time as compared to arhinitis therapeutic comprising BNP having the tail part.

Such effects of the external preparation of the present invention onrhinitis are exhibited within 8 minutes in general, preferably within 5minutes, more preferably within 3 minutes after its application.

Also the effect of the external preparation of the present invention onrhinitis as described above lasts for 4 hours or more in general,preferably for 8 hours or more, more preferably for 24 hours or moreafter it started to effect.

When the external preparation of the present invention is used as arhinitis therapeutic and/or prophylactic, applicable rhinitis includessuch as, without being particularly limited, for example, infectiousrhinitis including acute rhinitis and chronic rhinitis; hypersensitivenon-infectious rhinitis including combined (flower hypersensitivity)rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematousrhinitis and dry-nose type rhinitis; irritant-induced rhinitis includingphysical rhinitis, chemical rhinitis and radiation rhinitis; andatrophic rhinitis and idiopathic granulomatous rhinitis; and sinusitissuch as acute sinusitis, chronic sinusitis (maxillary empyema),eosinophilic sinusitis and paranasal cavity mycosis, The externalpreparation can be used for the purpose of treating or preventing oneore more of the above.

Combined rhinitis includes, for example, allergic rhinitis includingperennial allergic rhinitis and seasonal allergic rhinitis, andnonallergic rhinitis including vasomotor (essential) rhinitis andeosinophilic rhinitis.

Rhinitis with rhinorrhea includes, for example, gustatory rhinitis, coldair-induced rhinitis and senile rhinitis.

Congestive rhinitis includes, for example, drug-induced rhinitis,psychogenic rhinitis, gestational rhinitis, endocrine rhinitis and coldrhinitis.

Edematous rhinitis includes, for example, aspirin-hypersensitiverhinitis.

Among those mentioned above, the external preparation of the presentinvention exerts an excellent effect particularly on hypersensitivenon-infectious rhinitis, irritant-induced rhinitis and sinusitis,preferably on hypersensitive non-infectious rhinitis and chronicsinusitis, more preferably on combined (flower hypersensitivity)rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematousrhinitis and dry-nose type rhinitis, chronic sinusitis. Therefore, theexternal preparation of the present invention can be used for thepurpose of treating or preventing at least one or more rhinitis selectedfrom the above-mentioned group.

In terms of symptoms, since the external preparation of the presentinvention acts as described above, it can be used for any of rhinitiswith sneezing/rhinorrhea, rhinitis with nasal obstruction and rhinitiswith both symptoms.

In addition, the external preparation of the present invention exhibitsan effect of improving rhinitis which cannot be cured by a rhinitistherapeutic that has conventionally been used such as steroid drug.

(6) Other External Preparations

The external preparation of the present invention can also be used foran application other than those described above for the effect of thecyclic peptide as described above. In this case, the externalpreparation of the present invention can set an objective that is notthe effects of the cyclic peptide as described above. In this case, thecyclic peptide of the invention, a derivative and/or a pharmaceuticallyacceptable salt thereof is used for the purpose of assisting the maineffect of the external preparation or adding another effect to the maineffect.

Such application include such as, without being particularly limited,for example, body powder, deodorant, depilation agent, soap, bodyshampoo, bath agent, hand soap, perfume, sunscreen, and antiinflammatoryagent and antifungal agent.

2.2 Formulation

The external preparation of the present invention can exert the effectof its active ingredient, i.e., the cyclic peptide or a derivativeand/or pharmaceutically acceptable salt thereof, certainly and quicklyin the vicinity of the applied site by being locally applied to the siteof the interest (e.g., affected site) on skin or mucosa.

Such external preparation can be, without being particularly limited,for example, an agent for integument, eye drop, ear drop, nasal drop,buccal agent or suppository. Among these, when the external preparationof the present invention is a dermatitis therapeutic, a dermatitisprophylactic, an antipruritic, a skin-care product, an alopeciatherapeutic, an alopecia prophylactic, a hair growing agent or a hairgrowth stimulant, it is preferably an agent for integument. On the otherhand, when the external preparation of the present invention is arhinitis therapeutic and/or prophylactic, it is preferably a nasal drop.Furthermore, when it is a therapeutic/prophylactic of a corneal disease,it is preferably an eye drop.

When the external preparation of the present invention is an agent forintegument, it can be, without being particularly limited, for example,an external solid formulation, an external liquid formulation, a sprayformulation, an ointment, an emulsion, a cream, a gel formulation or apatch.

An external solid formulation is a solid formulation for applying orspraying onto such as skin. Such an external solid formulation includes,for example, a powder form external formulation.

An external liquid formulation is a liquid formulation for applying ontosuch as skin. Such an external liquid formulation includes, for example,a lotion and liniment.

A spray formulation is a formulation for spraying an active ingredientin a mist, powder, foam or paste form onto skin. Such spray formulationincludes, for example, an external aerosol and a pump spray formulation.

An ointment is a semi-solid formulation which is applied to skin andcomprises an active ingredient dissolved or dispersed in a base. Theointment can also be a lip cream for locally applying to lips, etc.

A cream is a semi-solid formulation which is applied to skin andemulsified as either oil-in-water or water-in-oil type.

A gel formulation is a gelled formulation which is applied to skin. Thegel formulation includes, for example, aqueous gel formulation andoil-based gel formulation.

A patch is a formulation which is attached to skin. The patch includes,for example, a tape or plaster.

A nasal drop is a formulation which is administered to nasal cavity ornasal mucosa. The nasal drop includes, for example, nasal powderformulation and a nasal drop. Among these, nasal drop is preferred.

In any formulation described above, the external preparation of thepresent invention comprises the cyclic peptide of the present inventionand/or a derivative thereof and/or a pharmaceutically acceptable saltthereof as described above.

When the formulation is an external liquid formulation, an ointment, acream or a gel formulation, the external preparation of the presentinvention comprises the cyclic peptide and/or a derivative thereofand/or a pharmaceutically acceptable salt thereof at a concentration of,for example, 0.0001 to 1000000 μg/g, preferably 0.001 to 10000 μg/g,more preferably 0.01 to 1000 μg/g, yet more preferably 0.1 to 100 μg/g.In another embodiment, it may comprises the cyclic peptide at aconcentration of 1 to 800 μg/g or 3 to 500 μg/g.

When the formulation is a spray formulation, the external preparation ofthe present invention comprises in the stock solution of the sprayformulation the cyclic peptide and/or a derivative thereof and/or apharmaceutically acceptable salt thereof at a concentration of, forexample, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, 0.01to 1000 μg/mL, more preferably 0.1 to 100 μg/mL, yet more preferably 1to 100 μg/mL. In another embodiment, it may comprises the cyclic peptideat a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

When the formulation is a patch, the external preparation of the presentinvention comprises the cyclic peptide and/or a derivative thereofand/or a pharmaceutically acceptable salt thereof at a concentration of,for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL,more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, itmay comprises the cyclic peptide at a concentration of 1 to 800 μg/mL or3 to 500 μg/mL.

When the formulation is a nasal drop, especially a liquid nasal drop,the external preparation of the present invention comprises in the nasaldrop solution (nasal drop) the cyclic peptide and/or a derivativethereof and/or a pharmaceutically acceptable salt thereof at aconcentration of, for example, 0.0001 to 1000000 μg/mL, preferably 0.001to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In anotherembodiment, it may comprises the cyclic peptide at a concentration of 1to 800 μg/mL or 3 to 500 μg/mL.

The external preparation of the present invention can be formulated, forany formulation as described above, by using methods and constituentmaterials known to those skilled in the art.

Available constituent materials include such as, without beingparticularly limited, for example, a gelator, oily ingredient, higheralcohol, fatty acid, ultraviolet absorbing agent, ultraviolet scatteringagent, powder, pigment, surfactant, polyhydric alcohol/sugar, polymer,bioactive ingredient, solvent, antioxidant, flavor and antiseptic agent.

Various organic or inorganic gelator compounds can be used.

An inorganic gelator compound includes such as, for example, hydrous orwater-absorbable silicate, for example, aluminum silicate (e.g.,bentonite), magnesium-aluminum silicate and colloidal silica.

As an organic gelator compound, a natural, semisynthetic or syntheticpolymer can be used. natural and semisynthetic polymers include such as,for example, polysaccharides such as cellulose, starch, tragacanth, gumarabic, xanthane gum, agar, gelatin, alginic acid and a salt thereof(e.g., sodium alginate and a derivative thereof), lower alkylcellulose(e.g., methylcellulose or ethylcellulose), carboxy- or hydroxy-loweralkylcellulose (e.g., carboxymethylcellulose or hydroxypropylcellulose).A synthetic polymer includes such as, for example, carboxylvinylpolymer, sodium polyacrylate, (vinylmethyl ether/ethyl maleate)copolymer, polymethacrylate, polyvinyl alcohol, polyvinylpyrrolidone,polyacrylate or polymethacrylate. Alternatively, as a gel formulation,commercially available gelators such as, for example, Lubrajel® NP,Lubrajel® CG, Lubrajel® DV, Lubrajel® MS, Lubrajel® OIL, Lubrajel® TW,Lubrajel® DS (Ashland Inc.) can also be used.

As an oily ingredient, for example, various ester, ether, hydrocarbon,silicone and fluorine oil phase ingredient, as well as animal and plantoils and a hardened oil thereof, and waxes of natural origin.

The ester oil phase ingredients include such as, for example, glyceryltri(2-ethyl hexanoate), cetyl 2-ethylhexanoate, isopropyl myristate,butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate,isocetyl isostearate, butyl stearate, butyl myristate, ethyl linoleate,isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearylmyristate, isostearyl palmitate, octyldocecyl myristate, isocetylisostearate, diethyl sebacate, diisopropyl adipate, isoalkylneopentanoate, glyceryl tri(capryl-caprinate), trimethylol propanetri(2-ethylhexanoate), trimethylol propane triisostearate,pentaerythritol tetra(2-ethylhexaonate), cetyl caprylate, decyl laurate,hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate,stearyl stearate, decyl oleate, cetyl ricinoleate, isocetyl myristate,isostearyl myristate, isocetyl palmitate, isostearyl palmitate, octylstearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate,octyldodecyl linoleate, isopropyl isostearate, cetostearyl2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethyleneglycol dioctanoate, ethylene glycol dioleate, propylene glycoldicaprinate, propylene glycol di(capryl-caprinate), propylene glycoldicaprylate, neopentyl glycol dicaprinate, neopentyl glycol dioctanoate,glyceryl tricaprylate, glyceryl triundecanoate, glyceryltriisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate,isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate,octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate,octyldecyl isostearate, polyglycerol oleic acid ester, polyglycerolisostearic acid ester, dipropyl carbonate, dialkyl (C12-18) carbonate,triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryllactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethylcitrate, acetyltriethyl citrate, acetyltributyl citrate, trioctylcitrate, diisostearyl malate, 2-ethylhexyl hydroxystearate,di(2-ethylhexyl) succinate, diisobutyl adipate, diisopropyl sebacate,dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate,cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryloleate, phytosteryl isostearate, phytosteryl oleate, isocetyl12-stearoylhydroxystearate, stearyl 12-stearoylhydroxystearate andisostearyl 12-stearoylhydroxystearate.

Hydrocarbon oil phase ingredients include such as, for example,squalane, liquid paraffin, α-olefin oligomer, isoparaffin, ceresin,paraffin, liquid isoparaffin, solid paraffin, polybutene,microcrystalline wax and vaseline.

Silicone oil phase ingredients include such as, for example,dimethylpolysiloxane, methylphenylpolysiloxane, methylcyclopolysiloxane,octamethylpolysiloxane, dacamethylpolysiloxane,dodecamethylcyclosiloxane, methyl hydrogen polysiloxane,polyether-denatured organo(polysiloxane),dimethylsiloxane-methylcetyloxysiloxane copolymer,dimethylsiloxane/methylstearoxysiloxane copolymer, alkyl-denaturedorgano(polysiloxane), terminal-denatured organo(polysiloxane),amino-denatured silicone oil, amino-denatured organo(polysiloxane),dimethiconol, silicone gel, acryl silicone, trimethyl siloxysilicate,silicone RTV gum.

Fluorine oil phase ingredients include such as, for example,perfluoropolyether, fluorine-denatured organo(polysiloxane), pitchfluoride, fluorocarbon, fluoroalcohol and fluoroalkyl-polyoxyalkyleneco-denatured organo(polysiloxane).

Animal and plant oils and a hardened oil thereof, and waxes of naturalorigin include such as, for example, animal and plant oils and thehardened oil thereof such as beef tallow, hardened beef tallow, lard,hardened lard, horse oil, mink oil, orange roughy oil, fish oil,hardened fish oil, egg yolk, jojoba oil; plant oils and a hardened oilthereof such as avocado oil, almond oil, olive oil, cacao butter,apricot kernel oil, kukui nut oil, sesame oil, wheat germ oil, rice germoil, rice bran oil, safflower oil, shea butter, soybean oil, eveningprimrose oil, camellia oil, corn oil, rapeseed oil, hardened rapeseedoil, palm kernel oil, hardened palm kernel oil, palm oil, hardened palmoil, peanut oil, hardened peanut oil, castor oil, hardened castor oil,sunflower oil, grape seed oil, jojoba oil, hardened jojoba oil,macadamia nut oil, meadowfoam oil, cottonseed oil, hardened cottonseedoil, palm oil, hardened palm oil, rose hip oil; waxes such as beeswax,beeswax having high acid value, lanolin, reduced lanolin, hardenedlanolin, liquid lanolin, carnauba wax and montan wax.

Higher alcohols include such as, for example, lauryl alcohol, myristylalcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, oleylalcohol, behenyl alcohol, 2-ethyl hexanol, hexadecyl alcohol, octyldodecanol.

Fatty acids include such as, for example, caprylic acid, capric acid,undecylenic acid, lauric acid, myristic acid, palmitic acid, palmitoleicacid, stearic acid, isostearic acid, oleic acid, linoleic acid,linolenic acid, arachic acid, arachidonic acid, behenic acid, erucicacid, 2-ethyl hexanoic acid.

Ultraviolet absorbing agents include such as, for example,paraminobenzoate, amyl paraminobenzoate, ethyl dihydroxypropylparaminobenzoate, glyceryl paraminobenzoate, ethyl paraminobenzoate,octyl paraminobenzoate, octyl dimethyl paraminobenzoate, ethylene glycolsalicylate, octyl salicylate, triethanol aminesalicylate, phenylsalicylate, butyl phenyl salicylate, benzyl salicylate, menthylsalicylate, benzyl cinnamate, octyl paramethoxy cinnamate, 2-ethylhexylparamethoxy cinnamate, glyceryl diparamethoxy cinnamatemono(2-ethylhexanoate), isopropyl paramethoxy cinnamate,paramethoxyhydrocinnamate diethanol amine salt, diisopropyl-disopropylcinnamate ester mixture, urocanate, urocanate ethyl, hydroxymethoxybenzophenone, hydroxymethoxy benzophenone sulfonate and a salt thereof,dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenonesulfonate, dihydroxybenzophenone, dihydroxy dimethoxybenzophenone,hydroxyoctoxybenzophenone, tetrahydroxybenzophenone, butylmethoxy-dibenzoyl methan,2,4,6-trianilino-p-(carbo-2-ethylhexyl-1-oxy)-1,3,5-triazine,2-(2-hydroxy-5-methylphenyl) benzotriazole, methyl-O-aminobenzoate,2-ethylhexyl-2-cyano-3,3-diphenyl acrylate, phenylbenzimidazole sulfate,3-(4-methyl benzylidene) camphor, isopropyl dibenzoylmethan,4-(3,4-dimethoxyphenylmethylene)-2,5-dioxo-1-imidazolidine propionate2-ethylhexyl, and polymer derivatives or silane derivative thereof,titanium oxide and zinc oxide and dispersion thereof. The zinc oxide andtitanium oxide may be surface-treated.

Dermal absorption auxiliary agents include such as, for example, aceticacid, sodium acetate, limonene, menthol, salicylic acid, hyaluronicacid, oleic acid, N,N-diethyl-m-toluamide (N,N-diethyl-3-methylbenzamide), n-butyl stearate, benzyl alcohol, isopropyl myristate,isopropyl palmitate, polypropylene glycol, crotamiton, diethyl sebacate,N-methylpyrrolidone, N-ethylpyrrolidone and lauryl alcohol.

Powders and pigments include such as, for example, pigments such asPigment Red 104, Pigment Red 201, Pigment Yellow 4, Pigment Blue 1,Pigment Black 401, basic dyes, HC colors, disperse dyes, direct colors,lake pigments such as Pigment Yellow 4 AL lake, Pigment Yellow 203 BAlake; polymers such as nylon powder, silk powder, urethane powder,silicone powder, methyl polymethacrylate powder, cellulose powder,starch, silicone elastomer spherical powder and polyethylene powder;colored pigments such as yellow iron oxide, red iron oxide, black ironoxide, chromic oxide, carbon black, ultramarine, iron blue; whitepigments such zinc oxide, titanium oxide, cerium oxide; extender pigmentsuch as tare, mica, sericite, kaolin and tabular barium sulfate; pearlpigment such as titanium mica; metal salt such as barium sulfate,calcium carbonate, magnesium carbonate, aluminum silicate and magnesiumsilicate; inorganic powder such as silica, alumina; metal soap such asaluminum stearate, magnesium stearate, zinc palmitate, zinc myristate,magnesium myristate, zinc laurate, zinc undecylenate; bentonite,smectite, boron nitride, etc. The shape (spherical, rod-like,needle-like, tabular, amorphous, scaly, spindle-shaped) and particlediameter of these powders are not particularly limited.

These powders and pigments may be pre-treated by known conventionalsurface processing such as, for example, fluorine compound processing,silicone processing, silicone resin processing, pendant processing,processing with silane coupling agent, processing with titanium couplingagent, oil solution processing, N-acylated lysine processing,polyacrylic acid processing, metal soap processing, amino acidprocessing, lecithin processing, inorganic compound processing, plasmaprocessing and mechanochemical processing.

As the surfactant, any of an anionic surfactant, a cationic surfactant,ampholytic surfactant and non-ionic surfactant can be used asappropriate.

Anionic surfactants include such as, for example, fatty acid soap,α-acylsulfonate, alkylsulfonate, alkylallylsulfonate,alkylnaphthalenesulfonate, alkylsulfate, POE alkylethersulfate,alkylamidesulfate, alkylphosphate, POE alkylphosphate,alkylamidephosphate, alkyloylalkyl taurine salt, N-acylamino acid salt,POE alkylether carboxylate, alkylsulfosuccinate, sodiumalkylsulfoacetate, acylisethionate, acylated hydrolyzed collagen peptidesalt and perfluoroalkyl phosphate ester.

Cationic surfactants include such as, for example,alkyltrimethylammonium chloride, stearyltrimethylammonium chloride,stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride,distearyldimethylammonium chloride, stearyldimethylbenzylammoniumchloride, behenyltrimethylammonium bromide, benzalkonium chloride,propyldimethylhydroxypropylammonium behenic amide chloride,diethylaminoethyl-stearamide, dimethylaminopropyl-stearamide and lanolinderivative quaternary ammonium salt. Cationic surfactants also includetertiary amines such as fatty acid amide dialkyl amine and saltsthereof.

Ampholytic surfactants include various ampholytic surfactants, forexample, those of carboxybetain type, amidebetain type, sulfobetaintype, hydroxysulfobetain type, amidesulfobetain type, phoshobetain type,aminocarboxylate type, imidazoline derivative type and amideamine type.

Non-ionic surfactants include such as, for example, propylene glycolfatty acid ester, glycerin fatty acid ester, polyglycerin fatty acidester, sorbitan fatty acid ester, POE sorbitan fatty acid ester, POEsorbitol fatty acid ester, POE glycerin fatty acid ester, POE alkylether, POE fatty acid ester, POE hardened castor oil, POE castor oil,POE-POP copolymer, POE-POP alkyl ether, polyether-denatured siliconealkanolamide laurate, alkylamine oxide, hydrogenated soybeanphospholipid, hydrogenized soybean phospholipid, polymer surfactant andbiosurfactant.

Natural surfactants may also be used, including such as, for example,lecithin, saponin and saccharide surfactant.

Polyhydric alcohols and sugars include such as, for example, ethyleneglycol, diethylene glycol, polyethylene glycol, propylene glycol,dipropylene glycol, polypropylene glycol, glycerin, diglycerin,polyglycerin, 3-methyl-1,3-butanediol, 1,3-butylene glycol, sorbitol,mannitol, raffinose, erythritol, glucose, sucrose, fructose, xylitol,lactose, maltose, maltitol, trehalose, alkylated trehalose, mixedisomerized sugar, sulfated trehalose and pullulan. Chemicallymodificated forms of these can also be used.

Polymers include such as, for example, anionic polymer compounds such asacrylate ester/methacrylate ester copolymer, vinyl acetate/crotonatecopolymer, vinyl acetate/crotonate/vinyl neodecanoate copolymer,methylvinyl ether maleate half ester, t-butyl acrylate/ethylacrylate/methacrylate copolymer, vinylpyrrolidone/vinyl acetate/vinylpropionate copolymer, vinyl acetate/crotonatecopolymer (RUBISET CA:BASF), vinyl acetate/crotonate/vinylpyrrolidone copolymer,vinylpyrrolidone/acrylate copolymer, acrylate/acrylamide copolymer,vinyl acetate/butyl maleate/isoisobornyl acrylate copolymer,carboxyvinyl polymer, acrylate/methacrylate alkyl copolymer, andampholytic acetate of dialkyl aminoethyl methacrylate polymer,ampholytic polymer compound such as octyl acrylamideacrylate/hydroxypropyl acrylate/butyl methacrylateaminoethyl copolymer,quaternized compound of vinylpyrrolidone/dimethylaminoethylmethacrylate, cationic polymer compounds such as methylvinyl imidazoliumchloride/vinylpyrrolidone copolymer, non-ionic polymer compounds suchsas polyvinylpyrrolidone, vinylpyrrolidone/vinyl acetate copolymer,vinylpyrrolidone/dimethylaminoethyl methacrylate copolymer,vinylcaprolactam/vinylpyrrolidone/dimethylaminoethyl methacrylatecopolymer. Polymer compounds of natural origin such as cellulose orderivatives thereof, keratin and collagen or derivatives thereof,calcium alginate, pullulan, agar, gelatin, tamarind seedpolysaccharides, xanthan gum, carrageenan, high-methoxyl pectin, lowmethoxyl pectin, guar gum, gum arabic, crystalline cellulose, arabinogalactan, can be karaya, gum tragacanth, alginates, albumin, casein,curdlan, gellan gum and dextran, and glucooligosaccharide, fucosecontaining polysaccharide, rhamnose containing polysaccharide can alsosuitably be combined.

Bioactive ingredients include substances which provide skin with somebioactivity when being applied to skin. Bioactive ingredients includethose with such as, for example, skin-lightening, anti-inflammatory,anti-aging, ultraviolet protection, slimming, firming, antioxidation,hair growth stimulating/hair growing, suppressing hair growth,moisturizing, promoting circulation, antimicrobial/sterilization,cool/warm feeling, promoting wound cure, alleviating irritation,analgesic and cell-activating effects. Bioactive ingredients includesuch as plant extracts, seaweed extracts, vitamins and derivativesthereof, amino acids, various peptides other than the cyclic peptide,biopolymers such as sodium hyaluronate and mucopolysaccharide,intercellular lipid constituents such as ceramide, phytosphingosine,cholesterol and phytosterol, and analogues thereof, and enzymaticingredients.

Examples of suitable combining ingredient include such as, for example,Angelica keiskei extract, avocado extract, sweet Hydrangea leaf extract,althea extract, arnica extract, aloe extract, apricot extract, apricotkernel extract, isoflavones, ginkgo extract, fennel extract, turmericextract, oolong tea extract, Rose Fruit extract, echinacea extract,Baikal skullcup extract, Amur Corktree extract, Coptis japonica extract,barley extract, Hypericum erectum extract, Lamium album extract, cressextract, orange extract, cacao extract, desiccated sea water, seaweedextract, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed silk,pumpkin seed extract, chamomile extract, carrot extract, Artemisiacapillaris extract, licorice extract, hibiscus extract, Pyracanthafortuneana extract, kiwi extract, cinchona extract, cucumber extract,guanosine, gardenia extract, Sasa veitchii extract, Sophora flavescensextract, cranberry extract, walnut extract, grapefruit extract, clematisextract, chlorella extract, mulberry extract, gentian extract, teaextract, yeast extract, burdock extract, rice bran fermentation extract,rice germ oil, comfrey extract, collagen, cowberry extract, Asarumsieboldii Miq. extract, bupleurum extract, umbilical cord extract,salvia extract, soapwort extract, bamboo grass extract, crataegusextract, zanthoxylum extract, shiitake mushroom extract, Rehmannia rootextract, Lithospermum root extract, Perilla frutescens extract, Tiliajaponica (Miq.) extract, Filipendula multijuga extract, Paeonialactillora extract, calamus extract, white birch extract, Equisetumarvense extract, Hedera helix extract, Crataegus oxyacantha extract,European elder extract, yarrow extract, peppermint extract, sageextract, tree mallow extract, cnidium extract, extract of Swertiajaponica (Schult.) Makino, soybean extract, jujube extract, soybeanfermentation extract, thyme extract, tea extract, clove extract, cogonextract, orange peel extract, Oenothera tetraptera extract, Centellaasiatica extract, Terminalia sericea extract, dong quai extract, commonmarigold extract, peach kernel extract, bitter orange peel extract,extract of Houttuynia cordata Thunb., tomato extract, natto extract,ginseng extract, garlic extract, wild rose extract, hibiscus extract,Ophiopogon japonicus extract, parsley extract, honey, banana flowerextract, hamamelis extract, parietaria extract, Isodon japonicus (Burm.)Hara extract, bisabolol, loquat extract, coltsfoot extract, butterburflower extract, Poria Sclerotium extract, butcher's-broom extract, grapeextract, propolis, luffa extract, safflower extract, peppermint extract,linden extract, tree peonyextract, hop extract, pine extract, horsechestnut extract, Lysichiton camtschatcense (L.) Schott extract,Sapindus mukurossi Gaertn. extract, Melissa extract, peach extract,bluebottle extract, eucalyptus extract, Saxifraga stolonifera extract,Citrus junos extract, coix seed extract, mugwort extract, lavenderextract, apple extract, lytchee (Litchi) extract, lettuce extract, lemonextract, Chinese milk vetch extract, rose extract, rosemary extract,Roman chamomile extract and royal jelly extract.

Examples also include such as biopolymers such as deoxyribonucleic acid,mucopolysaccharides, sodium hyaluronate, sodium chondroitin sulfate,collagen, elastin, chitin, chitosan and hydrolyzed eggshell membrane;moisturizing ingredients such as amino acids, hydrolyzed peptides,sodium lactate, urea, sodium carbonate pyrrolidone, betain, whey,trimethyl glycine, polypeptides such as lysine/arginine condensate etc.;intercellular lipid constituents such as sphingolipid, ceramide,phytosphingosine, cholesterol, cholesterol derivative, phytosterolderivative, phospholipid and analogues thereof; anti-inflammatory agentssuch as ε-aminoaminocaproic acid, glycyrrhizic acid, β-glycyrrhetinicacid, lysozyme chloride, guaiazulen, hydrocortisone, tea tree oil,vitamins such as vitamin A and derivatives thereof, vitamin B2 andderivatives thereof, vitamin B6 and derivatives thereof, vitamin C andderivatives thereof, vitamin D and derivatives thereof, vitamin E andderivatives thereof, calcium pantothenate, biotin and nicotinic acid;active agents such as allantoin, diisopropylamine dichloroacetate,4-aminomethylcyclohexane carbonate; antioxidants such as tocopherol,carotenoids, flavonoids, tannin, lignan and saponin; cell activatingagents such as α-hydroxy acid and β-hydroxy acid; circulationaccelerating agents such as γ-oryzanol and vitamin E derivative; woundcuring agents such as retinol and retinol derivative; skin-lighteningagents such as arbutin, kojic acid, placenta extract, sulfur, ellagicacid, linolenic acid, tranexamic acid, glutathione; hair growing agentssuch as cepharanthine, glycyrrhiza extract, capsicum tincture,hinokitiol, garlic extract iodide, pyridoxine hydrochloride,DL-α-tocopherol, DL-α-tocopherol acetate, nicotinic ac id, nicotinicacid derivative, calcium pantothenate, D-pantothenyl alcohol, acetylpantothenyl ethyl ether, biotin, allantoin, isopropylmethylphenol,estradiol, ethinylestradiol, carpronium chloride, Benzalkonium chloride,diphenhydramine hydrochloride, Takanal, camphor, salicylic acid,vanillyl nonylate amide, vanillyl nonanoate amide, piroctone olamine,glyceryl pentadecanoate, L-menthol, mononitroguaiacol, resorcin,γ-aminobutyric acid, benzethonium chloride, mexiletine hydrochloride,auxin, female hormones, cantharides tincture, Cyclosporine, zincpyrithione, hydrocortisone, minoxidil, polyoxyethylene sorbitanmonostearate, peppermint oil, Sasanishiki extract.

Antioxidants include such as, for example, sodium bisulfite, sodiumsulfite, erythorbic acid, sodium erythorbate, dilauryl thiodipropionate,tocopherol, tolyl biguanide, nordihydroguaiaretinoic acid,parahydroxyanisole, butyl hydroxyanisole, dibutyl hydroxytoluene,ascorbyl stearate, ascorbyl palmitate, octyl gallate, propyl gallate,carotenoids, flavonoids, tannin, lignans, saponins and plant extracts inwhich the antioxidative effect is recognized such as apple extract orclove extract.

Solvents include such as physiological saline, purified water, ethanol,lower alcohols, ethers, LPG, fluorocarbons, N-methylpyrrolidone,fluoroalcohols, volatile straight chain silicones and next-generationchlorofluorocarbons.

The cyclic peptide of the invention and/or a derivative thereof and/or apharmaceutically acceptable salt thereof can be used for preparation ofthe external preparation as described above, and therefore the presentinvention also relates to a use of the cyclic peptide of the inventionand/or a derivative thereof and/or a pharmaceutically acceptable saltthereof for preparing the external preparation.

3. Method of Using the External Preparation

Next, methods of using the external preparation of the invention isexplained.

A method of using the external preparation of the invention comprisesapplying the external preparation of the present invention as describedabove to the skin and/or mucosa of a subject.

Subjects include human and vertebrates such as, without beingparticularly limited, for example, birds and mammals. Animalsspecifically include, for example, experimental animals includingrodents such as mice, rats, gerbils, hamsters and guinea pigs, domesticanimals such as pigs, goats, horses, sheep and minks, pet animals suchas dogs and cats, primates such as human, monkeys, cynomolgus monkeys,rhesus monkeys, marmosets, orangutans and chimpanzees. On the otherhand, human may be excluded from the subjects.

Skin and/or mucosa of a subject to which the external preparation is tobe applied may be skin or mucosa at any site of the subject, and theexternal preparation is applicable, for example, to skin or mucosa ofhead (scalp), face, neck, arms, torso, arms, hands or feet, etc.

Specific method for using the external preparation of the presentinvention is as follows.

(1) Method of Treating and Preventing Dermatitis

When the external preparation of the present invention is used as atherapeutic/prophylactic of dermatitis, the external preparation can beapplied directly to the aimed site of skin and/or mucosa (e.g., theaffected site where dermatitis has been developed).

The application frequency is, without being particularly limited, forexample, 1 to 10 times a day, preferably 1 to 5 times a day, yet morepreferably 1 to 3 times a day. Because the external preparation of thepresent invention has a long lasting time, it exerts a sufficient effecteven if it is applied at relatively low frequency, for example, once aday.

In terms of dosage, the total amount of the cyclic peptide of theinvention and a derivative thereof and a pharmaceutically acceptablesalt thereof for one application can be, without being particularlylimited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to100 μg/mL, particularly preferably 1 to 100 μg/mL. In anotherembodiment, each dose may comprise the cyclic peptide of the inventionat a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

(2) Method of Alleviating or Resolving Itch

When the external preparation of the present invention is used as anantipruritic, the external preparation can be applied directly to theaimed site of skin and/or mucosa.

The application frequency is, without being particularly limited, forexample, 1 to 10 times a day, preferably 1 to 5 times a day, yet morepreferably 1 to 3 times a day.

In terms of dosage, the total amount of the cyclic peptide of theinvention and a derivative thereof and a pharmaceutically acceptablesalt thereof for one application can be, without being particularlylimited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to100 μg/mL, particularly preferably 1 to 100 μg/mL. In anotherembodiment, each dose may comprise the cyclic peptide of the inventionat a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

(3) Method of Using as a Cosmetic

When the external preparation of the present invention is used as acosmetic, the external preparation can be applied directly to the aimedsite of skin and/or mucosa.

The application frequency is, without being particularly limited, forexample, 1 to 10 times a day, preferably 1 to 5 times a day, yet morepreferably 1 to 3 times a day.

In terms of dosage, the total amount of the cyclic peptide of theinvention and a derivative thereof and a pharmaceutically acceptablesalt thereof for one application can be, without being particularlylimited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to100 μg/mL, particularly preferably 1 to 100 μg/mL. In anotherembodiment, each dose may comprise the cyclic peptide of the inventionat a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

(4) Method of Treating and Preventing Alopecia, and Method ofStimulating Hair Growth and Method of Growing Hair

When the external preparation of the present invention is used as atherapeutic/prophylactic of alopecia, a hair growth stimulant or a hairgrowing agent, the external preparation can be applied directly to theaimed site (e.g., decalvant site of scalp or skin)

The application frequency is, without being particularly limited, forexample, 1 to 10 times a day, preferably 1 to 5 times a day, yet morepreferably 1 to 3 times a day.

In terms of dosage, the total amount of the cyclic peptide of theinvention and a derivative thereof and a pharmaceutically acceptablesalt thereof for one application can be, without being particularlylimited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to100 μg/mL, particularly preferably 1 to 100 μg/mL. In anotherembodiment, each dose may comprise the cyclic peptide of the inventionat a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

(5) Method of Treating and Preventing Rhinitis

When the external preparation of the present invention is used as atherapeutic/prophylactic of rhinitis, the external preparation can beapplied directly to the aimed site (e.g., nasal cavity mucosa).

The application frequency is, without being particularly limited, forexample, 1 to 10 times a day, preferably 1 to 5 times a day, yet morepreferably 1 to 3 times a day.

In terms of dosage, without being particularly limited, for example, forone application to each nasal cavity, the total amount of the cyclicpeptide of the invention and a derivative thereof and a pharmaceuticallyacceptable salt thereof can be 0.0001 to 1000000 μg/mL, preferably 0.001to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In anotherembodiment, each dose may comprise the cyclic peptide of the inventionat a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

4. Medicament

Next, the medicament of the present invention is explained.

The medicament of the present invention comprises one or more cyclicpeptides of the invention or derivatives thereof or pharmaceuticallyacceptable salts thereof.

As mentioned above, similarly to BNP, the cyclic peptide of the presentinvention or a derivative thereof or a pharmaceutically acceptable saltthereof binds to the receptor NPR-A (also known as GC-A), which has aguanylate cyclase domain, and promotes the production of cyclicguanosine monophosphate (cGMP). It is considered to have effects suchas, for example, diuretic action, vasodilation, renin-aldosteronesecretion suppressing action, sympatholytic activity and hypertrophysuppressing action. Also, the compounds of the invention is consideredto have a superior efficacy and effect, particularly a superiorimmediate effect, as compared with BNP.

Therefore, the medicament of the present invention can be used for asimilar object as a conventional medicament comprising BNP as an activeagent.

Diseases for which the medicament of the present invention is applicableinclude such as, without being particularly limited, for example, theaforementioned various diseases as well as hypertension, unstableangina, acute myocardial infarction, edematous diseases, renal failure,cardiac failure, immune diseases, obesity and metabolic syndrome.

A formulation of the medicament of the present invention can be, withoutbeing particularly limited, addingly to the external preparations asmentioned above, for example, formulations for oral administration suchas tablets, capsules, granules, powders, oral solution, syrup and oraljelly, formulations for oral application such as buccal tablets, buccalspray formulation, buccal semi-solid formulation and mouthwash,formulations for administration via injection such as an injection andinfusion, dialysis formulation such as dialysis solution, as well as aninhalant, eye drop, ocular ointment, ear drop, vaginal tablet andvaginal suppository.

Working Examples

Hereinbelow, the present invention is further specifically illustratedby working examples. It should be noted that the present invention isnot limited by these working examples.

1. Preparation of the Cyclic Peptide

Firstly, a cyclic peptide composed of the amino acid sequence expressedby Formula I-a is synthesized.

Specifically, a linear peptide consisting of 17 amino acids was formedby sequentially binding amino acids by solid-phase peptide synthesisusing a peptide synthesizer. Subsequently, protecting groups at Cys1 andCys17 were detached before treating with iodine (I2) to form a cysteinebinding between oxidatively same amino acid residues, thereby forming acyclic peptide.

A composition comprising the obtained cyclic peptide was purified byreverse-phase high performance liquid chromatography (reverse-phaseHPLC) and then lyophilized to yield a purified cyclic peptide as whitepowder.

Mass spectroscopy was performed on the obtained cyclic peptide.

Conditions for HPLC are shown below:

Apparatus: Agilent 1100

Flow rate: 1.0 ml/minEluent A: 0.1% trifluoroacetic acid/waterEluent B: 0.1% trifluoroacetic acid/acetonitrileGradient: 80% Eluent B, isocratic

Conditions for Mass spectroscopy (MS) are shown below:

Apparatus: Thermo Finnigan LCQ Advantage

Ionization method: electrospray ionizationAnalytical method: ion trapping

The observed results, m/z=901.83 ([M+2H]²⁺), m/z=1801.84 ([M+H]⁺),confirmed that above peptide is in agreement with the theoretical valuesof the molecular weight (1802.07) and the mass number (1800.8069)calculated from the composition formula of the cyclic peptide ofinterest (C₇₂H₁₂₀N₂₄O₂₄S₃).

Furthermore, the purity of the above peptide was measured by HPLC usingfollowing conditions:

Column: Discovery C18, 4.6 mm×250 mm, particle diameter 5 micron

Column temperature: room temperature

Eluent A: 0.1% trifluoroacetic acid/water

Eluent B: 0.1% trifluoroacetic acid/acetonitrile

Gradient: 10 to 30% Eluent B/20 minutes

Flow rate: 1.2 ml/min

Temperature: room temperature

Injected volume: 20 μl

Detector: UV detector (detection wavelength: 215 nm)

The result of measurement confirmed that the purity of the obtainedprotein was 99.2%.

2. Preparation of Formulation 2.1 Preparation of Gel Formulation

Preparation of a gel-based formulation comprising a cyclic peptidecomposed of the amino acid sequence expressed by Formula I-a (in theworking examples below, “B ring” is referred to mean a cyclic peptideexpressed by such Formula I-a) (B ring gel formulation, WorkingExamples) and a gel-based formulation comprising human BNP (BNP gelformulation, Comparative Examples) were carried out as follows.

0.1 g of methyl-p-hydroxybenzoate (trade name: Mekkins-M, Ueno FineChemicals Industry, Ltd.), 0.2 g of phenoxyethanol and 3.0 g of1,2-pentanediol were weighed into one same vessel, heated to 60 to 70°C. to make a homogenous solution, and this solution was poured into amixer.

Next, added into the mixer 6.0 g of concentrated glycerin, and then themixture of 0.44 g of carboxyvinyl polymer (trade name: Carbopol® 940,Lubrizol Advanced Materials Corporation) and 0.08 g of xanthane gum(trade name: KELTROL® T, CP Kelco, Inc.), and the mixuter was stirredwith a paddle until they dispersed sufficiently.

Then 83.95 g of purified water was gradually added with stirring with apaddle. The mixer was heated to 70 to 80° C. while stirring with apaddle or disper until the dispersed contents were dissolved to give asolution. Subsequently, the disper was stopped and the solution wascooled immediately after confirming that the contents in the solutionwere dissolved. When the temperature of the mixer reached approximately40° C., 6.0 g of Lubrajel® NP from Ashland Inc. (glycerin 2.7 g,carboxyvinyl polymer 0.06 g, sodium polyacrylate 0.018 g, water 3.222 g)was added to the solution, mixed uniformly with a paddle. Subsequently,0.230 g of potassium hydroxide was further added to neutralize thesolution, then the rotation of the paddle was stopped when thetemperature of the mixer reached 25° C. to prepare a gel base.

Next, 20.1 mg of the the cyclic peptide (B ring) composed of the aminoacid sequence expressed by Formula I-a was dissolved in 144 mL ofphysiological saline to obtain B ring solution. 0.131 mL of thissolution was admixed with 10 g of the gel base obtained as describedabove, and the mixture was stirred uniformly to prepare a gel basedformulation (B ring gel formulation) containing B ring at aconcentration of about 1 μM (about 1.8 μg/g). Similarly, gel basedformulations containing B ring at concentrations of about 0.3 μM (about0.54 μg/g), about 0.5 μM (about 0.9 μg/g) and about 2.0 μM (about 3.6μg/g) were prepared. In the working examples below, as long as it isspecifically described otherwise, “B ring gel formulation” usedcontained B ring at a concentration of about 1 μM.

Next, 20.5 mg of human BNP-32 (American Peptide Company) was dissolvedin 118 mL of physiological saline to obtain BNP solution. 0.2 mL of thissolution was admixed with 10 g of the gel base obtained as describedabove, and the mixture was stirred uniformly to prepare a gel basedformulation (BNP gel formulation) containing BNP-32 at a concentrationof about 1 μM. Similarly, gel based formulations containing BNP-32 atconcentrations of about 0.5 μM and 2.0 μM were prepared. In the workingexamples below, as long as being specifically described otherwise, “BNPgel formulation” used contained BNP at a concentration of about 1 μM.

2.2 Preparation of Nasal Drop

A nasal drop comprising the cyclic peptide (B ring) composed of theamino acid sequence expressed by Formula I-a (B ring nasal drop, WorkingExamples) and a nasal drop comprising human BNP (BNP nasal drop,Comparative Examples) were prepared as follows.

Firstly, the cyclic peptide (B ring) composed of the amino acid sequenceexpressed by Formula I-a was dissolved in physiological saline, theconcentration was adjusted to prepare B ring nasal drop containing Bring at a concentration of about 1 μmol/1 (about 1.8 μg/g).

Next, B ring nasal drop was filled in a quantitative nasal spraycontainer (AS ONE Corporation) and the amount to be sprayed for eachadministration was adjusted to 100 μl (0.1 ml), resulting B ring nasaldrop.

Similarly, a BNP nasal drop comprising human BNP (American PeptideCompany) at a concentration of 1 μmol/1 was obtained, filled in aquantitative nasal spray container (AS ONE Corporation), and the amountto be sprayed for each administration was adjusted to 100 μl (0.1 ml),resulting BNP nasal drop.

3. Confirming Therapeutic Effect on Dermatitis 3.1 Confirming the Effectof B Ring Gel Formulation

For subjects suffering various dermatitis, B ring gel formulation asdescribed was applied onto the affected site, and changes in symptomsbefore and after the application were observed. When possible, as acomparative example, BNP gel formulation as described was applied ontoanother affected site on the same subject where B ring gel formulationhad not been applied, and changes in symptoms before and after theapplication were observed. For pruritus, VAS (Visual Analogue Scale) wasused for evaluating each affected site on 10 stages.

The examination results are shown in Tables 1 to 7 along with age,sexuality, symptoms of the subject, and formulation given to thesubject.

TABLE 1 Pruritus before and after Pre-application symptomsPost-application symptoms application Case Treatment Disease (severity)(severity) (VAS) Notes D1 2 μM atopic Face: presented with Face: achesremoved 2 min after 1 face 8→0 Age: 30's B ring gel dermatitis erythema,scales and skin application. Desiccation, scales and right arm 7→0 Sex:male formulation desiccation (moderate erythema

 After 3 days of 1 application disease). application, erythema, scales,

Limbs: presented with and desiccation remarkably remitted erythema,lichenification, (insignificant). skin desiccation and Right forearm:itches stopped to some scratch scar (severe). extent after 1.5 min on Bring-applied site. No itching or tingling after 2 min.

 remitteed after 4 min. Desiccation improved. Skin moisturized and soft,with natural appearance. No subjective discomfort (moderate). 2 μM Face:

 realized after 4 min. face 8→4 BNP gel erythema and scales moredistinguished left arm 7→4 formulation than B ring side. 1 applicationLeft forearm: after 4 min, skin was still dry. No improvement inlichenification. Rough and stiff skin (severe). D2 1 μM atopic Flushing,infiltrating erythema, Right back: itches stopped after 3 min. 8→0 Age:20's B ring gel dermatitis scales, many scratch scars Flushing erythema,infiltration and Sex: male formulation erythroderma (severe) accompaniedby scales remitted. Skin became soft 1 application strong itches causingsleep (moderate). 1 μM disruption and desire to Left back: still ichingafter 3 min. 8→4 BNP gel scratch. no remission of flushing erythema,formulation infiltration and scales (severe). 1 application D3 0.5 μMatopic Presented with infiltrating Right back: no tingling itches 30 sec6→0 Age 10's B ring gel dermatitis erythema, scales, papules, afterapplication. Skin felt normal. Sex: male formulation (moderate)accompanied by Erythema, infiltration and scales 1 application tinglingitches. remitted after 4 min. (mild). 0.5 μM Left back: still itchingafter 4 min, 6→6 BNP gel with no remission of erythema, formulationinfiltration and scales (moderate). 1 application

indicates data missing or illegible when filed

TABLE 2 Pruritus before and after Pre-application symptomsPost-application symptoms application Case Treatment Disease (severity)(severity) (VAS) Notes D4 2 μM atopic Presented with infiltrating Rightback: itches relieved just after 10→0 Age: 20's B ring gel dermatitiserythema, scales, many application. No itches after 2 min with Sex: maleformulation scratch scars with exudation remission of erythema andinfiltration 1 application (severe) accompanied by (moderate). strongitches that cause sleep Forehead: itches reduced after 1.5 min.disruption on the back, After 5 min skin was soft with nolichenification, erythema, lichenification. Erythema and scales scalesand scratch scars on remitted. Wound quickly healed. Scratch forehead(severe). scars epithelized. 2 μM Back: presented with many Left back:still itching after 4 min with no 10→3 BNP gel scratch scars withexudation, remission of erythema and infiltration formulationinfiltration, erythema, (severe) 1 application crusts (severe).Forehead: no improvement in lichenification, desiccation and scales,with remaining itches (severe) D5 1 μM atopic Presented withinfiltrating Right face: itches, edema and infiltration 9→0 Age: 20's Bring gel dermatitis erythema, edema and crusts remitted/relieved justafter application. Sex: male formulation (severe) accompanied by Skinfelt no itch and moisturized after 1 1 application strong tinglingithces. min (moderate). Back: presented with many No itches at all after2 min. Edema and scratch scars with exudation, erythema remitted. Righteye became infiltration, erythema, crusts easy to open. 1 μM and papules(severe). Left face: still itching after 3 min with no 9→4 BNP gelremission of erythema, infiltration and formulation edema. Beingdifficult to open left eye 1 application (severe). D6 1 μM atopicPresented with lichenification, Right back: itches improved/relieved 6→0Age: 10's B ring gel dermatitis

, erythema, after 3 min. Lichenification,

Sex: male formulation

scales, papules and many erythema, scales, scratch 1 application scratchscars (severe) scars all improved as compared to left. accompanied bystrong itches. Skin was soft and moisturized Back: presented with many(moderate) 1 μM scratch scars with exudation, Left back: still itchingafter 4 min with no 6→3 BNP gel infiltration, erythema, crusts remissionof erythema and infiltration formulation and papules (severe). (severe)1 application Forehead: lichenification, desiccation and scales notimproved, with itches left (severe).

indicates data missing or illegible when filed

TABLE 3 Pruritus before and after Pre-application symptomsPost-application symptoms application Case Treatment Disease (severity)(severity) (VAS) Notes D7 0.3 μM atopic Presented with infiltrating Noitches after 1 min. Erythema 10→0 Intractable Age: 20's B ring geldermatitis erythema, papules, exudation remitted and exudation stoppedstroid- Sex: male formulation and many scratch scars with after 10 min.1 application/day resistance 1 application crust (severe) accompaniedfor 3 days cleared of infiltrating by strong itches that cause erythema,papules, exudation and sleep disruption. scratch scars. Remarkablyimproved crusts, partly left mild erythema (mild). D8 0.5 μM atopicPresented with erythema, 5 days after application, erythema, 8→1Intractable Sex: male B ring gel dermatitis infiltration, scales,infiltration, scales, crusts and steroid- formulation crusts, scratchscars and wound quickly cured with little resistance 1 application, skindesiccation (severe). scratch scars left (mild). 5 days Desiccationremarkably remitted Skin moisturized and soft. D9 B ring gel atopic Leftforearm presented with Itches removed in 2 min (VAS 0). 10→0 Age: 30'sformulation dermatitis many scratch scars with Erythema and exudationimproved. Sex: male 2 applications/ exudation, infiltration, Severity ofrash improved to day 1 day erythema, papules, moderate disease levelafter accompanied with tingling 5 min. After 1 more application strongitches (VAS10) given on the same night, (severe) there was no itches for3 days thereafter. Scratch scars epithelized. D10 1 μM atopic Presentedwith infiltrating Right forearm: itches topped after 8→0 Age: 50's Bring gel dermatitis erythema, scales, papules and 3 min. Erythema andinfiltration Sex: female formulation many scratch scars (severe)improved (moderate). 1 application accompanied by intermediate 1 μMstrong itches Left forearm: still itching after 8→4 BNP gel 4 min withno remission of erythema formulation and infiltration (severe) 1application D11 1 μM atopic Presented with infiltrating Right face:immediately after 10→0 Age: 40's B ring gel dermatitis erythema, scalesand many application, itches and skin tautness Sex: female formulationscratch scars (severe) relieved, with erythema getting better. 1application accompanied by drastic No itching after 1 min. Erythema,itches and tautness. infiltration and desiccation Back: presented withmany remitted (moderate). 1 μM scratch scars with exudation, Left face:still itching after 3 min, 10→4 BNP gel infiltration, erythema, havingerythema, infiltration and edema formulation crusts and papules(severe). with swollen feeling (severe). 1 application

TABLE 4 Pruritus before and after Pre-application symptomsPost-application symptoms application Case Treatment Disease (severity)(severity) (VAS) Notes D12 1 μm atopic Strong tingling itches, Rightface: itches remitted after 3 min. 10 → 1 Age: 40's B ring geldermititis disrupted sleep, infiltrating Edema, erythema andinfiltration started Sex: formulation erythema, scales, exudation, to beremitted. After 30 min, anti- female 1 application cracks, crusts,scratch scars; inflammatory effect observed, exudation eyes cannot beopened due to stopped, wound epithelization promoted, exudate sealingeyelids cracks almost epithelized. Edema, (severe). erythema andinfiltration remitted to some extent. Eyelids became openable steadily.1 μm Left face: still having tingling itches after 10 → 6 BNP gel 3 minwith no remission of erythema and formulation infiltration. Itches tillleft after 30 min. 1 application D13 1 μm atopic Obvious infiltration,erythema Right face: erythema started to be 8 → 0 Age: 20's B ring geldermititis and papules all over the face, remitted and itches removedjust after (immediate Sex: formulation accompanied with strongapplication. Erythema and infiltration after female 1 application itchesand hot flash (severe). remitted after 4 min (moderate). application) 1μm Left face: itching with hot flush and 8 → 0 BNP gel redness justafter application. Itches (after 4 formulation removed after 4 min.minutes) 1 application D14 1 μm atopic Hand eczema of which main Righthand: itches removed after 3 min 9 → 0 Steroid- Age: 20's B ring geldermititis symptom is contact dermititis (VAS0). Lichenificationimproved, skin resistance; Sex: formulation due to detergent and gotsoft, erythema and infiltration intractable female 1 applicationdisinfectant. Presented with remitted (moderate). lichenification 1 μmlichenification, erythema, Left hand: no improvement in 9 → 4 whichcannot BNP gel scales and scratch scars lichenification and erythemaafter 10 min be remitted formulation (severe). Sleep disruption by(severe). within this 1 application strong itches (VAS9). short time (3min) by conventional therapy.

TABLE 5 Pruritus before and after Pre-application symptomsPost-application symptoms application Case Treatment Disease (severity)(severity) (VAS) Notes D15 1 μm atopic Presented with erythema,Erythema, infiltration, scales and crusts 9 → 1 Age: 20's B ring geldermititis infiltration, scales, crusts, wound epithelization improved 3days Sex: formulation scratch scars (severe), with after application.Scratch scars female 1 application/ strong itches. significantlyimproved. Only mild day erythema and scales observed (mild). 3 daysDesiccation relieved, improved and moisturzed skin texture. Itches andscratch scars significantly improved. D16 1 μm contact Contactdermititis due to hair Right side: tingling itches removed after 8 → 0Age: 60's B ring gel dermititis dye with erythena, edema 1 min. Itcheseliminated after 3 min Sex: formulation and infiltration along hairline(VAS0), felt better than left. Erythema female 1 application (moderatedisease) and infiltration remitted. accompanied by strongAnti-inflammatory effect observed. tingling itches (VAS8) Edema improved(mild). 1 μm Left side: symptoms less improved than 8 → 4 BNP gel right(moderate). formulation 1 application D17 1 μm chronic Presented withinfiltrating Right face: itches stopped just after 10 → 0 Age: 40's Bring gel eczema erythema, scales, application. Erythema remitted. NoSex: formulation lichenification, crusts, papules scales and desiccationafter 2 min. Skin female 1 application and skin desiccation (severe) wasmoisturized (moderate). accompanied by drastic itches Forehead: itchescontrolled after 1.5 min. and tautness. No lichenification after 5 min.Skin got soft. Erythema and scales remitted, scratch scars epithelizedand wound quickly healed. 1 μm Left face: still itching and taut after 4min 10 → 5 BNP gel with no remission of desiccation, scales, formulationerythema and infiltration (severe). 1 application D18 1 μm eczemaPresented with erythema, After 15 min, erythema, edema, 8 → 0 Age: Bring gel edema, infiltration, scales and infiltration and scalessignificantly Sex: formulation papules (severe). remitted and skintexture improved female 1 application (mild). 1 μm After 15 min, stillmany scales, 8 → 4 BNP gel erythema, edema and infiltration left.formulation Skin texture is rough and still itching. 1 application

TABLE 6 Pruritus before and after Pre-application symptomsPost-application symptoms application Case Treatment Disease (severity)(severity) (VAS) Notes D20 1 μm wheal, Presented with multiple Rightlower limb: itches removed just 10 → 0 Age: 20's B ring gel insect biteswheals, erythema and very after application. Erythema started to be Sex:formulation strong itches on both lower remitted after 1.5 min. Whealsfemale 1 application limbs. disappeared in about 1 hour. D31 1 μm wheal,Presented with multiple Left lower limb: itches removed 1 min 10 → 0Age: 30's B ring gel insect bites wheals and very strong itches afterapplication, wheals remitted after 2 Sex: formulation on right lowerlimb. min. female 1 application D21 1 μm childhood Presented withdesiccation, Right back: itches stopped after 30 sec. 8 → 0 Age: 10's Bring gel xerotic many milary large papules, Desiccation, scales anderythema Sex: formulation eczema desquamation, strong pruritus, improvedin a few minutes, Skin got female 2 applications/ and many scratch scarson moisturized. After 3 days, wounds day trunk; incrustation, erythemaquickly epithelized leaving one scratch 3 days and lichenification dueto scar. Skin is smooth and moisterized scratching (severe). with nodesiccation (insignificant). 1 μm Left back: desiccation, scales and 8 →3 BNP gel erythema left after few minutes. formulation 1 application D321 μm parapsoriasis Presented with multiple Scales, erythema andinfiltration remitted N/A Age: 40's B ring gel guttataerythrokeratoderma with after 4 min. Sex: formulation sticking smallscales on back. female 1 application D33 0.5 μm psoriasis Both lowerlimbs presented Scales and erythema started to be N/A Age: 60's B ringgel vulgaris with many red plaques with remitted 10 min after 1stapplication. Sex: male formulation attached small scales having After 1week, no scales left. 2 applications/ clear margin. day 1 week D34 2 μmpsoriasis Many red plaques with Back psoriasis: thick scales started tobe N/A Age: 30's B ring gel vulgaris attached thick scales havingremitted in 3 min after application, Sex: male formulation clear marginpresented on almost disappeared after 30 min. 1 application back andneck. Neck psoriasis: erythema and scales remitted in 3 min afterapplication.

TABLE 7 Pruritus before and after Pre-application symptomsPost-application symptoms application Case Treatment Disease (severity)(severity) (VAS) Notes D23 2 μm cyshidrotic Fingers presented with manyErythema and bullae remitted in 2 min N/A Intractable Age: 30's B ringgel eczema intractable bullae, being after application. After 1 day,erythema with Sex: male formulation dyshidrosis eczematous with erythemaand scales remitted and bullae steroid 2 applications/ and cracks.disappeared. ointment. day 1 day D24 2 μm miliaria Red small papules andflare Pruritus, erythema and red small N/A Age: 50's B ring gel hyper-with itches. papules remitted after 3 min. Sex: male formulationhidrosis 1 application D25 1 μm chronic Many wheals with drasticPruritus removed just after application. 10 → 0 Age: 50's B ring gelurticaria itches. Wheals started to be gradually remitted Sex: maleformulation just after application and disappeared 1 application after 7min. D26 1 μm rosacea Suffering from rosacea for 2 Tautness and tinglingitches removed N/A Intractable Age: 30's B ring gel years. 2 months offacial just after application. Diffuse flare, after 2 yrs of Sex:formulation application of a steroid follicular papule and pustuleremitted conventional female 1 application ointment exacerbated theafter 1 min. After 1 week of 2 therapy symptoms, developingapplication/day, anti-inflammatory effect without telangiectasia,diffuse flare, was observed. Diffuse flare, follicular satisfactoryfollicular papule and pustule papule and pustule remarkably therapeuticon cheeks. Subjective improved. Itches and hot flush removed. effect.symptoms include tingling itches and tautness. D35 4 μm perioral 2 yearsof facial application of Diffuse flare and follicular papule N/A Age:60's B ring gel dermititis a steroid ointment resulted in remarkablyremitted 2 min after Sex: formulation circumoral telangiectasia,application. After 2 application/day for 2 female 1 application diffuseflare and follicular days flare improved and scales papule. Subjectivesymptoms removed. include heat sensation.

TABLE 8 Pruritus before and after Pre-application symptomsPost-application symptoms application Case Treatment Disease (severity)(severity) (VAS) Notes D36 2 μm rosacea Presented with circumoralDiffuse flare and follicular papule Age: 50's B ring gel telangiectasia,diffuse flare remarkably remitted 2 min after Sex: male formulation andfollicular papule. application 1 application D37 1 μm acne Oily skinaccompanied by Seborrhea remitted 2 min after N/A Faster and Age: 20's Bring gel vulgaris suppurative inflammation with application.Anti-inflammatory effect stronger Sex: female formulation many pustulesand comedos remitted suppurative inflammation and anti- 1 application/on lower jaw. pustules on lower jaw. Pustules reduced inflammatory dayand dried 3 days after application, and effect than 3 days domedosdisappeared. applying/ administrating conventional antibiotics. D38Right cheek: acne Multiple follicular papules and Anti-inflammatoryeffect observed 3 min N/A Age: 40's 1 μm vulgaris pustules on bothcheek. Oily after application, pustules and papules Sex: female B ringgel skin. started to be reduced and flattened, and formulation flarestarted to be removed. After 1 1 application/ week pustules and papulessubstantially day remitted and flare further remitted. After 2 weeks 2weeks pustules, papules and flare almost disappeared. Left cheek: Nochange in symptoms 3 min after BNP gel application. After switching to Bring single formulation, similar effects as right application, cheekwere obtained. then 1 μm B ring gel formulation 1 application/ day 2weeks D39 Right cheek: acne Oily skin accompanied by Anti-inflammatoryeffect by B ring Age: 10's 2 μm vulgaris sppurative inflammation withapplication was observed on right cheek Sex: male B ring gel multiplered papules and in 3 min. Suppurative inflammation formulation pustuleson both cheeks. remitted. 1 application Left cheek: No anti-inflammatoryeffect on gel formulation gel-applied left side. 1 application D40 4 μmacne Seborrheic skin with multiple Comedos faded in 5 min after Age:10's B ring gel vulgaris comedos and follicular application. Oily skinimproved to better Sex: female formulation papules/pustules on forehead.texture. Red papules and pustules 1 application reduced.

As shown in Tables 1 to 8, in all subjects, or in all dermatitis of anysymptoms, emission or elimination of various symptoms of dermatitis wasobserved at the site where B ring gel formulation was applied.

Specifically, when B ring gel formulation was applied, remission orelimination of pruritus was observed immediately after the application.Generally in dermatitis, patients tend to scratch where pruritus wasfelt, potentially causing severer dermatitis. Such a remarkableremission or elimination of pruritus in this manner can consequentlyprevent aggravation of dermatitis.

In all cases, the effects of B ring gel formulation were superior tothose with BNP gel formulation. Specifically, B ring gel formulation hadfaster-acting and longer-lasting effects as well as more potentantipruritic effect as compared with BNP gel formulation. It wasconfirmed that these effect cannot be obtained by the gel base.

3.2 Comparing B Ring Gel Formulation to Gel Base by Two-SidedApplication

For subjects suffering various dermatitis, either B ring gel formulationor the gel base was applied to the affected site, and changes insymptoms before and after the application were observed (Table below).

TABLE 9 Case Applied site Treatment Disease Pre-application symptomsPost-application symptoms E1 Right B ring atopic Face presented withinfiltrating Flare and pigmentation removed 2.5 min after Age: 20'sdermatitis erythema, scales and application. Skin elasticity anddesiccation Sex: female pigmentation, with intractable improved. Itcheseliminated. Left gel base itches No remission of pigmentation,infiltrating erythema, scales and desiccation, with itches left. E2Right B ring chronic Stiffness and hard lichenification Flare remittedsoon after application. After Age: 30's eczema on both forearms, withinfiltrating 3 min skin softened and moisturized. Itches Sex: maleerythema and desiccation. eliminated. Left gel base No improvement inlichenification, itching and flare after 3 min. E3 Right B ring atopicPresented with hot flush and Flare started to fade soon afterapplication, Age: 10's dermatitis puffiness. and was remarkably remittedafter 3 min. Sex: female Left gel base No improvement in flushing after3 min. E4 Right B ring chronic Stiffness and hard lichenification Flareremitted soon after application, Age: 40's eczema on both forearms, withinfiltrating remarkably remitted after 3 min with improved Sex: maleerythema, scales and hot infiltration and hot sensation, and softenedsensation. skin. Left gel base Infiltration felt 3 min afterapplication. Skin was hard with unimproved flare and hot sensation.

4. Confirming Cosmetic Effect

Effects of a cosmetic comprising B ring were evaluated from the resultsobtained using a gel formulations, a bath agent and a hair cosmeticproduct (shampoo or rinse) or body soap comprising the B ring to confirmtheir effects.

4.1.1 Confirming Skin-Improving Effect (Comparing B Ring Gel Formulationto BNP Gel Formulation)

The B ring gel formulation or BNP gel formulation was applied tosubjects. The B ring gel formulation was applied on the right cheek oreye of each subject, the BNP gel formulation on the left cheek or eye,and changes in skin conditions were observed and compared (Table below).The changes in skin conditions were determined based on physical sensoryevaluation by the subject and our objective observation as physicians.

TABLE 10 Case Applied site: method Pre-application symptomsPost-application symptoms F1 Right: B ring Presented with wrinkles,cheek 2 min after application, wrinkles on eye corner and forehead wereAge: 69 gel formulation flabbiness, pigmentation on eye shallowed, skinwas full. After 3 min, cheek flabbiness and skin Sex: female 0.5 μmol,corner. texture were improved, and pigmentation on eye corner was less 1application prominent. After application for 3 days, large wrinkle oncheek was shallowed, flabbiness was improved, skin was firm, resilient,full and moisturized. Left: applied No improvement in wrinkles after 3min. gel base F2 Right: B ring Presented with wrinkles, cheek 2 minafter application, wrinkles at eye corner due to desiccation Age: 45 gelformulation flabbiness and desiccation. was less prominent, skin texturewas improved, moisturized and Sex: female 0.5 μmol, softened. 1application Left: applied No improvement in wrinkles and desiccationafter 2 min. gel base F3 Right: B ring Presented with desiccation, light30 sec after application, skin desiccation was improved, skin was Age:21 gel formulation erythema, tautness and itch. moisturized and flexiblewith no tautness. After 1 min, flare was Sex: female 2 μmol, remittedand itches were removed. After 4 min, further softness was 1 applicationgiven and desiccation was remarkably improved. Left: applied Noimprovement observed in desiccation, itching and flare after gel base 4min. F4 Right; B ring Presented with desiccation and 3 min afterapplication, skin desiccation was improved, skin was Age: 24 gelformulation light erythema. moisturized and firm, with improved texture.Skin was flexible, Sex: female 2 μmol, soft and moisturized. 1application Left: applied No improvement observed in desiccation andflare after 3 min. gel base F5 Right: B ring Presented with wrinkles onboth 2 min after application, wrinkles on both corners of eyes were lessAge: 28 gel formulation corners of eye, and desiccation. prominent,cheek dryness was improved, skin was moisturized and Sex: female 2 μmol,flexible. Perioral desiccation was remitted and skin became full 1application and flexible. Left: applied No improvement observed indesiccation and wrinkles after 2 min gel base at gel-applied site on theleft. F6 Right: B ring Presented with skin desiccation, 1 min afterapplication, itches were remarkably remitted. After Age: 30 gelformulation oily skin and red papules around 3 min, skin desiccation andflare were improved, and skin was Sex: female 2 μmol, nose, and lighterythema and moisturized and flexible with improved texture. Red papules1 application rough skin texture. around nose almost disappeared,combination state of desiccation and oily skin was improved, and skin iskept healthy. Left: applied 3 min after application, desiccation, flareand itches are left, gel base and seborrhea and papules around nose werenot remitted.

Table below summarizes examples in which B ring gel formulation wasapplied without applying two-sided. The changes in skin conditions weredetermined by similar method as described above.

TABLE 11 Case Method of application Pre-application symptomsPost-application symptoms G1 B ring gel formulation Presented with cheekflare, open After B ring application, flare was repressed, skin Age: 501 μmol pores, rough and hard skin texture. texture was improved, skinwas moisturized and softened. Sex: female 1 application G2 B ring gelformulation Presented with wrinkles and 3 min after application at Bring-applied site, wrinkles Age: 49 2 μmol desiccation on forehead andeye on forehead and eye corner were shallowed and less prominent, Sex:female 1 application corner. skin desiccation was improved, and skin wasmoisturized and flexible. G3 B ring get formulation Presented with maleskin 1 day after application at ring-applied site, skin desiccation Age:40's 1 μmol desiccation and roughness. and roughness were improved, andrazor burn was prevented. Sex: male 1 application G4 B ring gelformulation Presented with miliaria and 3 min after application at Bring-applied site, miliaria was Age: 50's 2 μmol pruritus accompaniedwith flare. prevented, flare and itches were remitted. Sex: male 1application

Accordingly, it was shown that B ring of the present invention has askin moisturizing effect and is capable of improving skin texture, andthat it is also effective in improving skin, for instance in lightening(spots, dullness) and anti-aging (flabbiness, resilience, largewrinkles) as well as in improving mucosal condition such as roughenedlips.

In all subjects, a remarkable improving effect in skin and itspersistence was observed at the site where B ring gel formulation hadbeen applied as compared to where BNP gel formulation had been applied.Specifically, effects as follows were observed at the site where B ringgel formulation had been applied as compared to where BNP gelformulation had been applied with significance: effects of improvingskin, moisturizing skin, improving skin texture, providing skin withresilience and firmness, softening skin, suppressing skin desiccation,fading fine wrinkles, supplying and keeping skin with water and oil,shallowing and diminishing crow's feet, eliminating skin rawness,eliminating and suppressing itches, relieving skin roughness andmaintaining skin condition healthy.

Particularly, in subjects in their 20's or 30's, remarkable effects suchas improving or eliminating wrinkles, improving cheek flabbiness, andsupplying and keeping skin with moisture, water and oil, and the effectssuch as giving skin firmness and tightness, lifting up cheeks,eliminating skin rawness and relieving skin roughness were observed atthe site where B ring gel formulation had been applied as compared tothe site where BNP gel formulation had been applied, and these effectswere greater as compared to those at BNP gel-applied site.

Moreover, in subjects in their 40's and 50's, remarkable effects such asimproving flabbiness, large wrinkles and desiccation, giving skinfirmness, fading dullness and spots were exhibited immediately after theapplication of B ring gel formulation, and these effects were greater ascompared to those upon applying BNP gel formulation.

The effects exhibited significantly faster in B ring gel formulation ascompared to BNP gel formulation. Specifically, effects were confirmed inmost cases such that wrinkles were vanished or started to vanish andfaded and skin was made full and resilient. These effects lasted for atleast several hours thereafter. No irritating symptoms were observedwith application of B ring gel formulation.

FIG. 1 shows a photograph of the right-side face of a subject in her60's after the application of B ring gel formulation. In this subject,effects were observed at 20 minutes after the application of B ring gelformulation such that the skin were moist, provided with moisture,complexion and softness, given firmness, improved texture, provided withresilience, and large wrinkles faded. This result was compared to BNPgel formulation as a comparative example, indicating that the cyclicpeptide of the invention is not only effective on fine wrinkles but alsois effective on wrinkles and flabbiness, and that it is also effectivein fading spots and dullness.

4.1.2 Confirming Skin-Improving Effect (Comparing B Ring Gel Formulationto Gel Base)

In order to examine the presence or absence of the contribution by thegel base to the effects of B ring gel formulation and BNP gelformulation, three subjects were administered B ring gel formulation ontheir right face and the gel base on the left, and changes in skinconditions on the subject's faces were observed (Table below).

TABLE 12 Case Applied site: method Pre-application symptomsPost-application symptoms H1 Right: B ring gel Rough, stiff and dry skintexture. After 2 min skin was left soft. Age: 48 formulation Sex: femaleLeft: applied gel base Skin was tingling. Open pores and rough skintexture were not improved. H2 Right: B ring Sensitive dry skin withroughness 1 min after application, skin tingling sensation Age: 26 andtingling stopped. After 2 min, B ring-applied side was felt Sex: femalemore moisturized. Left: applied gel base 1 min after application, skinwas still tingling. After 2 min, the subject left that moisturizingeffect is weak. H3 Right: Sensitive skin with tingling. Age: 30 Left:applied gel base Skin was tingling and temporarily red. Skin desiccationSex: female was not improved. White scales were observed.

As a result, all subjects felt tingling sensation at the site where thegel base was applied, and dry feeling of skin was not improved. Also, inall subjects, no effect (e.g., improved skin texture or closed pores)was observed at the site where the gel base was applied. On the otherhand, in all subjects, the skin area on which B ring gel formulation wasapplied has been moisturized and softened.

4.2 Observation on Mucosal Condition Upon Application of B Ring GelFormulation.

The B ring gel formulation was applied to normal subjects who hadsymptoms of roughened lips, and changes in mucosal condition wereobserved (Table below). The changes in mucosal conditions weredetermined based on physical sensory evaluation by the subject and ourobjective observation as physicians.

TABLE 13 Case Applied site: method Pre-application symptomsPos-application symptoms I1 B ring 1 μmol Cracks on mouth corner withpain. Skin became moisturized soon after application. After 2 min, Age:20's 1 application Desiccation and wrinkles of lips. cracks on mouthcorner were remitted. After 5 min, lips were Sex: female soft and nolonger felt dry or tingling. I2 B ring 0.5 μmol Annoying lipdesiccation. 3 min after application, lips became moisturized. Age: 30's1 application Sex: female I4 B ring 2 μmol Annoying lip desiccation.After 2 min, lips were full and soft, and wrinkles were less Age: 30's 1application prominent. Moisturized. Sex: female I3 B ring 2 μmol Lipswere dry and tingling, After 2 min, lips were full, soft andmoisturized, and wrinkles Age: 30's 1 application with wrinkles andscales were less prominent. Tingling sensation stopped. Sex: female I4Right: B ring Cracks on mouth corner with pain. 2 min after application,cracks on mouth corner were remitted Age: 20's 1 μmol Dry and roughenedlips with no more pain, and moisturized. Sex: male 1 application Left:gel base 2 min after application, cracks on mouth corner and pain were 1application not improved

Accordingly, it was confirmed that the cyclic peptide of the inventionhas an activity of improving mucosal condition and it also has animmediate effect in such activity.

4.3 Observation on Scalp/Hair Condition Upon Application of B Ring GelFormulation.

The B ring gel formulation was applied to normal subjects who hadsymptoms of scalp/hair, and changes in conditions of scalp and hair wereobserved (Table below). The changes in scalp/hair were determined basedon physical sensory evaluation by the subject and our objectiveobservation as physicians.

TABLE 14 Applied site: Case method Pre-application symptomsPost-application symptoms J1 B ring 0.5 μmol Presented with desiccationand After 2 min. itches were removed, scalp was relieved from Age: 40's1 application itches of head. roughness and desiccation and moisturized.After that scalp Sex: male was prevented from desiccation and keptmoisturized. J2 B ring 1 μmol Presented with erythema and itch Hairgrowth was stimulated, and an increase in hair volume Age: 50's 1application/day, of head, and thinning and falling of was observed, andthinning of hair was improved. Hair was Sex: female 3 weeks hair.provided with moisture, flexibility and radiance. J3 B ring 1 μmolPresented with thinning and falling A decrease in falling hair wasobserved, showing an effect Age: 40's 1 application/day, of hair. ofpreventing alopecia. Hair growth was stimulated, and an Sex: female 5days increase in hair volume was observed, and thinning of hair startedto be improved. Hair was provided with moisture, flexibility andradiance. J4 B ring 2 μmol Presented with seborrheic dandruff After 2min, itches were removed, scalp seborrhea and Age: 40's 1 applicationand itches. dandruff started to be improved. After that scalp becameSex: female and was kept clean. J5 B ring 2 μmol Presented withdesiccation, mild After 1 min. itches were removed. After 2 min, scalpAge: 20's 1 application erythema and itches. seborrhea and dandruffstarted to be improved. After 4 min, Sex: male erythema and skinroughness of scalp was improved. After that skin roughness wasprevented. J6 B ring 2 μmol Presented with scalp seborrhea, After 2 min,itches were removed, scalp seborrhea was improved. Age: 60's 1application dandruff and itches. After that dandruff was removed, scalpbecame healthy and clean. Sex: male J7 B ring 1 μmol From the beginningof 5th month of Upon being applied to alopecia 5 month post partum, aneffect Age: 30's To decalvant site 3rd pregnancy, significant hair losswas exhibited on hair loss in eyebrows and head where hair Sex: femaleon head and brows in eyebrows, lashes and scalp growth was stimulated,improving thinning of hair. of subject: 1 occurred, loosing almost allhair. application/day post partum, continued

Accordingly, BNP cyclic peptide of the present invention can provide andkeep scalp and hair with moisture, give them moderate water and oil,improve and prevent desiccation, and clean scalp and hair. It is furthereffective for suppressing itch and dandruff of scalp and also effectivefor preventing thinning or loosing hair, for growing hair and promotingor stimulating hair growth, and forcing hair growth, and furthermoreeffective for an improvement in alopecia after illness or postnatalalopecia.

4.4 Observation on Skin Condition Upon Using Bath Agent Comprising BRing

Subjects were given bath in 37 to 41° C. hot water comprising B ringdissolved at 0.01 μM (100 ml of 20 μM B ring solution dissolved in 200 Lhot water) once a day for 14 days, and skin condition of the subjectswas compared to the case of hot water alone (Table 14). The changes inskin conditions were determined based on physical sensory evaluation bythe subject and our objective observation as physicians.

TABLE 15 Pre-application symptoms (

, cracks, chaps Case Treatment and eczema) Post-application symptomsChanges in subjective symptoms K1 B ring Eczema Gradual remission ofitches after bath was remitted. Age: 30's addition erythema and skinSkin was moisturized. Sex: female desiccation. K2 Hot water Eczema Flareand desiccation Enhanced itches and desiccation Age: 30's exacerbated.after bath. Sex: female K3 BNP Eczema No remission of skin No subjectiveremssion of skin Age: 30's addition desiccation or erythema. desiccationat this concentration. Sex: female K4 B ring Chaps, rough skin Remissionof desiccation. Remission of itch and pain. Skin Age: 60's addition ofhands, itches became smooth Sex: female with tingling K5 Hot water Chapsand rough Exacerbation of desiccation. Exacerbation of itches and Age:60's skin of hands tingling after some time after Sex: female bath. K6 Bring

 and itches Remission of

Remission of itches and improvement Age: 20's addition in skin texture.Sex: male K7 Hot water

 and itches No improvement in

No remission of itches. Age: 20's Sex: male K8 B ring Cracks and painCracks gradually became Relief of pain. Skin was moisturized. Age: 40'saddition on sore shallower and improved Sex: female upon application K9Hot water Cracks and pain No improvement in cracks. No remission ofpain. Age: 40's on sore Sex: female

indicates data missing or illegible when filed

Accordingly, it was confirmed that the bath agent comprising B ring ofthe present invention has an ameliorating effect on miliaria, cracked orchapped skin, and also an improving effect on eczema. Furthermore, itwas shown to improve desiccation, pain and itch of the skin, indicatingthat it is effective for improvement of skin condition.

4.5 Observation on Conditions of Scalp and Hair or Skin Upon Using HairCosmetics (Shampoo, Rinse) or Body Soap Comprising B Ring 4.5.1 AShampoo Comprising B Ring was Mixed and Prepared in Composition asFollows.

TABLE 16 Name of ingredient (tradename) Content (%) A Sodiumcocoylmethyltaurine solution 10.0 (NIKKOL CMT-30) Sodium laureth lactate(NIKKOL SBL-2N-27) 20.0 Lauryl betaine solution (NIKKOL AM-301) 10.0Cocamide DEA 4.0 Antiseptic q.l. B Citric acid 0.1 Propylene glycol 2.0Guar hydroxypropyl trimonium chloride 0.5 Water to fill up 100.0 CWater, B ring (B ring concentration 1 μM) 1 mL

A and B were dissolved with heat at 70° C. To A, B was added, stirredand mixed. C was added at 40 to 35° C. while being further stirred, andallowed to cool to room temperature as kept being stirred.

4.5.2 A Conditioner Comprising B Ring was Mixed and Prepared inComposition as Follows.

TABLE 17 Name of ingredient (tradename) Content (%) A Pentylene glycol1.50 BG 3.50 Glycerin 1.00 Methyl paraben 0.20 Carbomer 0.20 EDTA-2Na0.10 Water to fill up 100.0 B Composite emulsifier (NIKKOL NikkomuleseLC) 4.00 Methyl heptyl laurate 3.50 Squalane 0.50 Cetearyl alcohol 1.50Macadamia nut oil 0.50 Avocado oil 0.50 Shea oil 0.50 Propyl paraben0.10 C Water, B ring (B ring concentration 1 μM) 1 mL

A and B were dissolved with heat at 80° C. Then A was stirred by ahomomixer while B was gradually added thereto, and the mixture wasemulsified. C was further added to the mixture, and the mixture wasallowed to cool to 35° C. as kept being stirred.

4.5.3 A Body Soap Comprising B Ring was Mixed and Prepared inComposition as Follows.

TABLE 18 Name of ingredient (tradename) Content (%) A Cocoyl glutamicacid TEA solution 30.0 Sodium trideceth-4 carboxylate 5.0 (KIKKOL ECTD-3NEX) Sodium cocoamphoacetate solution 10.0 (KIKKOL AM-101) PEG-50hydrogenated castor oil (KIKKOL HCO-50) 0.5 1,3-butylene glycol 5.0Antiseptic q.l. B EDTA-2Na q.l. Water to fill up 100.0 C Water, B ring(B ring concentration 1 μM) 1 mL

A and B were dissolved with heat at 80° C. Then B was added to A whilebeing stirred. C was further added to the mixture at 40 to 35° C. whilekept being stirred, and the mixture was allowed to cool to roomtemperature as kept being stirred.

Subjects used shampoo, rinse or body soap prepared as above once a dayfor 14 days, and resulted conditions of scalp and hair or skin of thesubjects were assessed (Table below). Changes in skin condition wasassessed by dandruff of scalp. Changes in erythema was assessed byvisual observation by the subject. Itches, moisturized feeling, combing,glow, resilience and elasticity of hair were assessed by subject'sphysical sensory evaluation.

TABLE 19 Case Treatment Pre-application symptoms Post-applicationsymptoms Subjective changes L1 shampoo/ Presented with erythema andRemission of scalp Improvement in itches. Hair was Age: 60's treatmentitches on scalp, and thinning erythema and a decrease moisturized andprovided with Sex: male and falling hair. in hair falling. resilience,radiance and elasticity. L2 shampoo Presented with desiccationImprovement in dandruff Improvement in itches. Hair was Age: 20's anddandruff on scalp and desiccation of scalp. moisturized and providedwith Sex: female accompanied with itches resilience, radiance andelasticity. Improved. With better combing. L3 body soap Dry andsensitive skin. Improvement in dry skin, improvement in itches afterbath. Age: 60's relief of itching after bath. Sex: female L4 body soapDry and sensitive skin. Improvement in dry skin, Improvement in itchesafter bath. Age: 60's relief of itching after bath. Sex: female L5 bodysoap Dry skin. Improvement in dry skin. Skin was moisturized after bath.Age: 20's Sex: male

Accordingly, the use of the hair agent comprising B ring of the presentinvention had an fast-acting improving effect on itch, erythema anddesiccation of scalp. Moreover, it was shown to be effective inimproving symptoms of thinning and falling hair when it was usedcontinuously for at least 2 weeks. Furthermore, it was shown that theuse of the body soap comprising B ring of the present invention has animproving effect on dry or sensitive skin.

5. Confirming Therapeutic Effect on Alopecia

Either B ring gel formulation or BNP gel formulation was applied, andtheir effects on symptoms associated to various alopecia were observed(Table below). The changes in symptoms were determined based on physicalsensory evaluation by the subject and our objective observation asphysicians.

TABLE 20 Case Treatment Disease Post-application symptoms A21 Right: Bring 1 μmol Male alopecia 7 weeks after application, hair became elasticand thick, Sex: male 1 application/ a drastic decrease in hair-fallingConsequently, hair became Age: 50's day, continued. dense and thinningless prominent B ring-containing gel Left: BNP gel formulation exhibitedmore remarkable hair growing affect formulation 1 application/ day,continued. A5 B ring gel Male alopecia and 2 weeks after startingapplying B gel formulation, the Sex: male formulation alopecia

existing soft hair in parietal area became thicker and Age: 50's 1application/ accompanied with longer and turned black.. Hair gotresilient and alestic. day onto head dandruff. Previous Newly grownterminal hairs remarkably decreased thinning decalvent site, use of

-containing area. Dandruff was suppressed overall, particularly incontinued. drug caused strong parietal area.

 was eliminated. irritation, itches and erythema to the subject and itsuse was terminated A22 B ring gel Seborrhoic alopecia 2 weeks afterapplication, thinning or hair was improved. Sex: female formulationaccompanied with exhibiting no more showing-through of scalp confirmingAge: 40's 2 application/ dandruff. hair-growing stimulation. Dandruffstopped, scalp seborrhea day onto was improved and pruritus was removed.Furthermore, scalp parietal and skin became clean. decalvant site.

indicates data missing or illegible when filed

TABLE 21 A10 B ring gel formulation Alopecia universalis. 2 weeks afterstarting application, on both sides of head, Sex: male 0.5 μmol Alopeciaon whole head. regeneration of pores and growth of terminal hair wereAge: 20's BNP gel formulation Previous therapy with confirmed, whichwere more remarkable and hair growth area 0.5 μmol carpronium chloridewas wider on the B ring gel formulation-applied right side. and steroidpulse first Hair growth area kept increasing to the whole head. Theexerted hair growth- density, elongation rate and thickness of hair weregreater stimulating effect, but on the B ring gel formulation-appliedright side. Upon caused complete hair increasing the concentration ofapplication to 1 μmol, loss after 1 month. rate of growth and elongationwere increased. A11 Right side of head decalvant Ophiasis and multipleRight forehead ophiasis site: 7 days after starting applying Sex: femalesite: B ring gel formulation alopecia. B ring gel formulation, a hairgrowth stimulation effect was Age: 40's 1 application/day for 1 week.observed and hair became thicker terminal hair, hair-growing 1 or 2applications/week for 1 area kept increasing. year thereafter. Left BNPgel formulation-applied site: few soft hair observed, Left side of headdecalvant whose growth was slow and in obviously smaller area. site: BNPgel formulation After continuing 1 or 2 applications/week for 1 year: 1application/day for 1 week. Right forehead B ring gelformulation-applied site: hair- 1 or 2 applications/week for 1 growingarea kept increasing and hair elongation rate was year thereafter. fast(FIG. 2). Left BNP gel formulation-applied site: hair growth wasobserved though its area was smaller as compared to the right side, andmajority of hairs was thin and soft (FIG. 2).

TABLE 22 A12 Head decalvant site: Alopecia areata 1 week afterapplication, remarkable stimulation and growth Sex: female B ring gelformulation of terminal hair was observed. Age: 20's 1 application/day,continued. A12 Subject's head decalvant Alopecia areata 1 week afterapplication, remarkable stimulation, growth Sex: female site: B ring gelformulation and elongation of terminal hair was observed. Age: 30's 1application/day, continued. A24 Right half of head decalvantDrug-induced Pre-application: 1 month after termination of anticancerSex: female site: B ring gel formulation alopecia agent therapy.Bilateral severe diffuse alopecia on whole Age: 50's (1 μmol), 2applications/ head. Thinning of hair on whole head. Hair was short andday for 2 weeks thin, mostly white. Left half of head decalvant 1 dayafter application at B ring-applied site, subjective site: BNP gelformulation hair elongation and growth stimulation were observed, and (1μmol), 2 applications/ grown hairs were predominantly black (non-white).After 2 day for 2 weeks weeks, hair growth was observed at both sides.Hair were elongated with less falling hairs. Hair elongation rate isfaster on B ring-applied site. Hairs are also thicker and denser, andcontain more non-white hairs as compared to BNP-applied site. 3 weeksafter application, remarkable growth of terminal hairs was observed.

5.2 Case Summary

In the cases of the subjects having female pattern alopecia, malepattern alopecia, alopecia universalis, ophiasis or alopecia areatamultilocularis as described above, a significant decrease in fallinghair was observed, and the area of terminal hair growth was expanded,and the growth of the terminal hair was also faster, when B ring gelformulation of the working example was applied compared with BNP gelformulation as a comparative control (See, FIG. 2). Addingly, theexisting hairs obtained resilience and their elasticity was increased.In all cases, symptoms were significantly improved as compared to thecases when BNP gel formulation of the comparative example was applied.Accordingly, B ring gel formulation had superior effects on the symptomsdescribed above as compared to BNP gel formulation.

In addition, the B ring gel formulation could improve seborrhea, cleanscalp and suppress dandruff. It also prevents hairs from turning white,grows black hairs, and improves thinning of hair. Moreover, it exhibitsan antipruritic effect within 10 minutes, demonstrating an excellentimmediate effect. It could suppress ithes within 3 minutes afterapplication in some cases.

6. Confirming Therapeutic Effect on Rhinitis 6.1 Cases

Either ring nasal drop, BNP gel nasal drop was sprayed, and theireffects on rhinitis in the subject was observed (Table 17). The changesin symptoms were determined based on physical sensory evaluation by thesubject and our objective observation as physicians.

TABLE 23 Case Treatment Disease Post-application symptoms (severity) R1Right nasal cavity: Perennial chronic Right nasal cavity (B ring nasaldrop-treated side): Subject felt breath goes through right Sex: female Bring nasal rhinitis and nasal cavity soon after application. After 30sec, air goes through nasal cavity. After 2 Age: 20's formulation 0.1ml, rhinorrhea. 20 or min. nose was clear and the subject could breathecomfortably. After 3 min, felt fresh 1 spray more blow/day and retainednasal discharge was removed. No discharge by blowing. A day after Leftnasal cavity: application, no discharge from right nasal cavity. BNPnasal Left nasal cavity (BNP nasal drop-treated side): 3 min afterapplication, the discharge formulation 0.1 ml, still retained in nasalcavity and drained by blowing. A day after application, mild nasal 1spray obstruction and rhinorrhea symptoms in left nasal cavity withsmall amount of retained discharge. R2 Right nasal cavity: Rhinitis withsevere Right nasal cavity (B ring nasal drop-treated side): Subject feltbreath goes through right Sex: female B ring nasal nasal obstructionnasal cavity soon after application. After 30 sec, obstruction wasimproved, subject's Age: 30's formulation 0.1 ml, and feeling heavy.suffering in breathing was reduced in right nasal cavity. After 1.5 min,suffering in 1 spray Right nasal cavity breathing was removed in rightnasal cavity and breath went though. A day after Left nasal cavity: hasseverer application, still no obstruction or rhinorrhea. Ths subjectfelt no irritation upon treatment BNP nasal obstruction symptom. inright nasal cavity formulation 0.1 ml, The subject feels dry Left nasalcavity (BNP nasal drop-treated side): 30 sec after application,obstruction of 1 spray and tingling by left nasal cavity was slightlyimproved, though the subject did not feel breath goes steroid nasaldrops. through as in the right cavity. A day after application,obstruction or rhinorrhea in left nasal cavity were removed. The subjectfelt mild dryness in left nasal cavity. R3 Right nasal cavity: Perennialallergic Right nasal cavity: Subject felt breath goes through nasalcavity 1 min after application. Sex: female B ring nasal rhinitis withAfter 3 min nasal obstruction was improved, the subject could breathethrough nose. Age: 20's formulation 0.1 ml, rhinorrhea and nasal After 5min the subject could breathe comfortably through nose, with no nasaldischarge 1 spray obstruction. by blowing. Right nasal cavity hasseverer obstruction with retention of nasal discharge.

TABLE 24 R4 Right nasal cavity: Chronic rhinitis with Right nasal cavity(B ring nasal drop-treated side): 1 min after treatment, nasal Sex:female B ring nasal severe rhinorrhea and obstruction was improved tosome extent. After 2 min, obstruction was improved, the Age: 30'sformulation 0.1 ml, nasal obstruction. subject could breathe throughnose as usual, and rhinorrhea stopped. After 3 min, no 1 sprayApplication of steroid nasal discharge from the right nasal cavity byblowing. Left nasal cavity: nasal drops could not Left nasal cavity (BNPnasal drop-treated side): 1 min after treatment, nasal discharge BNPnasal improve symptoms when was retained in the left nasal cavity withno improvement in obstruction After 2 min, formulation 0.1 ml, they weresevere. obstruction was improved to some extent, though discharge wasretained, showing 1 spray rhinorrhea symptom. After 3 min, nasaldischarge was not drained by blowing from left nasal cavity due toobstruction. After 8 min, nasal discharge was drained by blowing. R5Right nasal cavity: Allergic rhinitis with Right nasal cavity (B ringnasal drop-treated side): 30 sec after treatment, obstruction Sex:female B ring nasal rhinorrhea as main was improved and the subjectcould breathe through nose. After 2 min, the subject Age: 20'sformulation 0.1 ml, symptom and mild could easily breathe through nose.After 8 min, no nasal discharge was drained by 1 spray obstruction.blowing from the right nasal cavity. A day after treatment (after 26hours), nasal Left nasal cavity: discharge was controlled, and breathingwas easier through the right nasal cavity as BNP nasal compared to theleft. formulation 0.1 ml, Left nasal cavity (BNP nasal drop-treatedside): 2 min after treatment, obstruction was 1 spray still observed.After 8 min, small amount of discharge was drained by blowing. A dayafter treatment (after 26 hours), nasal discharge was controlled. R6Right nasal cavity: Allergic rhinitis Right nasal cavity (B ring nasaldrop-treated side): nasal obstruction was slightly Sex: female B ringnasal with both rhinorrhea improved soon after treatment. After 1 min,the subject could easily breathe though Age: 20's formulation 0.1 ml,and nasal obstruction nose. After 4 min, no nasal discharge was drainedby blowing from the right nasal cavity. 1 spray having constant Nasalobstruction was resolved. After 10 min, pruritus was removed. A dayafter Left nasal cavity: obstruction and treatment (after 28 hours), noobstruction or rhinorrhea was observed. BNP nasal severe rhinorrhea.Left nasal cavity (BNP nasal drop-treated side): 1 min after treatment,nasal obstruction formulation 0.1 ml, 10-20 blow/day. was not improved.After 3 min, obstruction was still not improved and rhinorrhea was 1spray Regular dose of oral observed. After 3 min, small amount ofdischarge was drained by blowing. After 10 min, anti-allergic drugobstruction was slightly improved but not resolved. After 2 hours,obstruction was did not sufficiently resolved and the subject couldbreathe though nose. A day after treatment (after 28 improve nasalhours), no obstruction or rhinorrhea was observed. obstruction andrhinorrhea. Steroid nasal spray causes mucosa pain.

TABLE 25 R21 Right nasal cavity: Rhinitis with continuous rhinorrheaRight nasal cavity (B ring nasal drop-treated side): Sex: female B ringnasal and sneeze. Nasal discharge was 30 sec after treatment, thesubject could easily breathe Age: 20's formulation 0.1 ml, drained byfacing down. through nose. After 1 min rhinorrhea stopped. No nasal 1spray discharge by facing down. R8 Right nasal cavity: Chronic allergicrhinitis (with both Right nasal cavity (B ring nasal drop-treated side):Sex: female B ring nasal rhinorrhea and obstruction). The 1 min aftertreatment, obstruction was improved and the Age: 20's formulation 0.1ml, subject regularly feels nasal subject could easily breathe throughnose. The subject 1 spray + 1 spray obstruction. Application of steroidfelt no irritation by treatment. The effect lasts for 1 20 min laternasal formulation causes the week thereafter, and rhinorrhea aridobstruction were Left nasal cavity: subject strong irritation.controlled. No nasal discharge by blowing. BNP nasal Accompanied withrhinorrhea and Commercial steroid nasal formulation caused a sharpformulation 0.1 ml, eye pruritus. irritation and recurrence of symptomswithin 2 hours. 1 spray + 1 spray Left nasal cavity (BNP nasaldrop-treated side): 20 min later R9 Right nasal cavity: Allergicrhinitis (with rhinorrhea). Right nasal cavity (B ring nasaldrop-treated side): Sex: female B ring nasal The subject has rhinorrheaall day 2 min after treatment, the subject could breathe through Age:30's formulation (1 μmol) and usually blows 20 or more times nose, withno irritation in the right nasal cavity. 0.1 ml, 1 spray a day.Application of steroid nasal After 4 min the subject could breathethrough nose, with Left nasal cavity: formulation causes the subject nonasal discharge drained. BNP nasal strong dry feeling on mucosae of Leftnasal cavity (BNP nasal drop-treated side): formulation (1 μmol) nasalcavity and pharynx. The 2 min after treatment, obstruction was improvedand the 0.1 ml, 1 spray effect of the steroid nasal subject couldbreathe through nose. After 4 min, in the formulation lasts only 3-4hours, left nasal cavity, the subject drained small amount of and afterthe effect ended, nasal discharge by blowing. In the right nasal cavity,sneezing and nasal discharge last the effect of the nasal treatmentlasted for 4 hours. for several hours and need Blowing causedobstruction in the left nasal cavity but not frequent blowing. in theright. The subject felt that B ring nasal formulation is much better tothe steroid nasal formulation regarding the effect on the symptoms,long-lasting and fast-acting effect, lower frequency of use required,and absence of irritating symptom upon use.

TABLE 26 R10 Right nasal cavity: Allergic rhinitis with rhinorrhea Rightnasal cavity (B ring nasal drop-treated side): Sex: male B ring nasaland obstruction complicated with 2 min after nasal treatment,obstruction was improved Age: 30's formulation (1 μmol) rhinosinusitis.Obstruction in the and the subject could breathe through nose. Thesubject 0.1 ml, 1 spray right nasal cavity due to could breathe morecomfortably in the right nasal cavity Left nasal cavity: rhinosinusitisis severer than the as compared to the left. The subject felt noirritation BNP nasal left and could not be improved by in the rightnasal cavity. After 4 min, the subject could formulation (1 μmol)steroid nasal formulation, making very easily breathe, with no nasaldischarge drained by 0.1 ml, 1 spray the subject incapable of breathingblowing. through nose. If left untreated, Left nasal cavity (BNP nasaldrop-treated side): nasal discharge is drained from 4 min after nasaltreatment, nasal obstruction was not nostrils spontaneously. resolved.Nasal discharge was decreased as compared to pre-treatment, thoughdraining was still observed. R11 Right nasal cavity: Perennial rhinitiswith rhinorrhea Right nasal cavity (B ring nasal drop-treated side):Sex: male B ring nasal and obstruction complicated with 2 min afternasal treatment, the subject could breathe Age: 20's formulation 0.1 ml,rhinosinusitis, having severe through nose. After 5 min, nasal dischargestopped, 1 spray obstruction. Very high frequency enabling the subjectto breathe though nose. The subject Left nasal cavity: of blowing. Thesesymptoms were felt no obstruction and was able to breathe through noseBNP nasal not improved by any previously throughout 1 week after nasaltreatment. formulation 0.1 ml, used nasal formulation. Left nasal cavity(BNP nasal drop-treated side): 1 spray 5 min after nasal treatment,obstruction was not resolved, with nasal discharge present in the leftnasal cavity. After 10 min. obstruction was not resolved, making thesubject incapable of breathing through nose.

6.2 Case Summary

In all cases described above, both rhinorrhea and nasal obstruction werequickly improved or eliminated when B ring nasal drop of the workingexample had been applied as compared to the case when BNP nasal drop ofcomparative example had been applied. Accordingly, B ring nasal drop hadfaster effect than BNP nasal drop. Addingly, the effect of B ring nasaldrop was more than equal to that of BNP nasal drop, and because theeffect lasted for longer time period, it can suppressing recurrence ofsymptoms.

The following table summarizes information from the cases describedabove, within the range recognizable at the time when symptoms of nasalobstruction and rhinorrhea were improved.

TABLE 27 No resolution within within within within 3 min 5 min 10 min 10min Resolution of rhinorrhea B ring nasal formulation 3 4 1 0 BNP nasalformulation 0 0 0 7 Resolution of nasal obstruction B ring nasalformulation 6 5 0 0 BNP nasal formulation 0 0 0 8

7. Analysis of Binding State of Human BNP and Type A Receptor

The superior pharmacological effects of the B ring-compound of theinvention on various diseases relative to conventional BNP has beenexplained so far with reference to data based on respective clinicalcases. Such pharmacological effects is also supported by the result ofthe conformation analysis of the compound performed by the inventors,which is explained by following experimental report for reference.

In order to investigate binding state of human BNP (BNP-32) and itsreceptor Type A receptor (NPR-A), an in silico analysis was performed byhomology modeling using conformation. Swiss-Pdb viewer and SWISS-MODELwere used for modeling.

7.1 Template Structure

Firstly, prior to the above analysis, a template structure forinvestigation on binding state of human BNP and Type A receptor wasselected. As this template structure, the conformation of the complex ofrat NPR-A and rat ANP peptide (PDB ID: 1T34) were used. Rat NPR-A is ahomodimer composed of A and B strands. The conformation of such ratNPR-A has been determined by X-ray crystalline structural analysis suchthat 21 residues from Cys7 to Arg 27 of rat ANP were bound. Theconformation of rat NPR-A was obtained from the database of proteinconformation, Protein Data Bank. Amino acid homology between human NPR-Aand rat NPR-A is 85%.

7.2 BNP Peptide Model

In the present study, as BNP peptide model, a region from Cys10 to Arg30in human BNP (BNP-32, SEQ ID NO: 13,Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His), i.e., an amino acid sequence (SEQ IDNO: 14,Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg)was used. In the peptide model above, the amino acid sequence of B ringof the invention corresponds to a region from Cys10 to Cys26 of humanBNP above, i.e.,Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys (SEQID NO: 15).

7.3 Homology Modeling

In order to speculate the amino acid residues that are involved inbinding of human NPR-A and human BNP, a complex model in which BNPpeptide is bound to human NPR-A was constructed by homology modeling.Specifically, human BNP peptide model was constructed based on thetemplate structure of rat ANP peptide, and human NPR-A model structurebased on the template structure of rat NPR-A.

Next, residues that are involved in the interaction was speculated fromthe amino acid residues detected between human BNP peptide model andhuman NPR-A.

The results showed that BNP peptide model is bound to human NPR-A dimerbeing sandwiched between the A strand and B strand. Amino acids in theamino acid sequence, e.g., the amino acid sequence used as the model(SEQ ID NO: 14,Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg)are expressed as “amino acid (number)”. Namely, the number inparentheses denotes the relative position counted from the N-terminal ofthe amino acid sequence, for example, Phe2 indicates an amino acid thatis the second Phe counted from the N-terminal of the peptide model.

In the constructed complex model, the presence of a hydrophobic bond byPhe2 side chain, a hydrogen bond by Phe2 main chain, and hydrogen bondsby side chains of Arg4, Met6, Arg8, Ser10 and Ser11 were speculatedbetween BNP peptide model and the A strand of NPR-A. Phe2, Arg4, Met6,Arg8, Ser10 and Ser11 in BNP peptide model correspond to Phe11, Arg13,Met15, Arg17, Ser19 and Ser20 in human BNP respectively. Namely, thisresult suggests that these amino acid residues in human BNP is likely tobe the residues that contribute to NPR-A activation.

7.4 Discussion

Thus, by in silico conformation analysis, it was speculated that, inhuman BNP, the amino acid residues that are present in its cyclic partare likely to contribute to NPR-A activation, whereas the amino acidresidues present in the tail part are not. It was also suggested thatBNP cyclic structure is a smaller molecule than BNP-32 and thereforecapable of binding easily and quickly. In fact, a clinical applicationof a peptide having BNP cyclic structure confirmed that BNP cyclicstructure provides faster therapeutic effect than BNP-32. This clinicalresult is consistent with that of the in silico analysis that BNP cyclicstructure contributes to NPR-A activation and can bind to it more easilyand quickly than BNP-32. Thus, BNP cyclic structure has a superiortherapeutic effect than a general BNP peptide, and therefore is adifferent substance.

On the other hand, an NMR analysis revealed that ANP does not take anyparticular conformation in a solution where its conformation greatlywobbles; this is considered to be similar for BNP. Namely, in human BNP,assuming that the amino acid residues in the cyclic part contribute toits binding to human NPR-A, it is speculated that the amino acidresidues in the tail part rather prevent human BNP from entering intothe narrow BNP binding site sandwiched between A and B strands of humanNPR-A due to its large wobbling. Therefore, it is considered that thecyclic part of human BNP is a relatively smaller molecule thanconventionally known human BNP, enabling itself to enter into BNPbinding site of NPR-A more easily and quickly. Moreover, it isspeculated that the the cyclic part of human BNP has a higher affinityto human NPR-A than BNP-32. This supports the clinical outcomesdescribed herein that the peptide having only the cyclic part of BNPexhibits therapeutic effect faster than BNP-32.

In order to speculate the effect of BNP cyclic structure (B ring) fromnon-human species on human Type A receptor, complex models of humanNPR-A and BNP rings from pig, bird or rat were generated to surmiseinteraction. The results suggested that a sufficient effect of theinvention can be expected by using BNP ring from non-human animalspecies (e.g., pigs, birds or rats), as long as it shows an affinity tohuman NPR-A.

8. Speculating Replaceable Amino Acid Residues in BNP Cyclic Moiety

Using the constructed model structure of the complex, replaceablity ofamino acid residues other than those considered to be involved in theinteraction was investigated.

Specifically, in order to investigate whether the peptide in which aminoacid residue that is not assumed to be involved in the interaction hasbeen replaced with another amino acid is capable of binding to NPR-A, amutant model of BNP was generated to analyze the interaction. Swiss-Pdbviewer was used for modeling.

Specifically, amino acid residues Gly12, Lys14, Asp16, Ile18, Ser21,Ser22, Gly23, Leu24 and Gly25 in human BNP were targeted for theinvestigation on their replaceability with other amino acids in terms offollowing points:

-   -   No steric hindrance caused by binding to NPR-A. No interatomic        collision observed in the model structure of NPR-A and BNP.    -   No influence on electrostatic potential on surface.    -   No large increase in intramolecular energy value (no unnatural        angle or twist caused in intermolecular binding).    -   No non-naturally occurring hydrogen bond formed between the        strands of NPR-A and BNP and within BNP strand.    -   No cavity (cavity, niche) formation.

Among those described above, intramolecular energy was calculated byComputeEnergy command of Swiss-Pdb viewer. Intramolecular energy wascalculated from the sum of the length of binding, bond angle, twist andbinding energy, etc. in the unit kilojoule/mol (Kj/mol).

The results of analysis indicated the presence of replaceable amino acidresidues in Gly12, Lys14, Ile18, Ser21, Ser22, Leu24 and Gly25 among theamino acid residues of human BNP (Table below).

TABLE 28 human BNP Replaceable amino acid Gly12 Ala, Val, Ser, Thr Lys14Arg Asp16 none Ile18 Val Ser21 Thr, Ala, Val, Gln, Leu, Ile, Met Ser22Thr, Ala, Val Gly23 none Leu24 Ala, Val, Ile, Met Gly25 Ala, Ser

Accordingly, even when the replacement of the amino acid correspondingto those described in the table above took place in the cyclic part ofhuman BNP, i.e., the peptide expressed by the Formula I-a, it wassuggested that the peptide replaced in such a way exerts a similareffect as the peptide composed of the amino acid sequence expressed byFormula I-a.

9. Confirming Therapeutic Effect of the Cyclic Peptide with Amino AcidReplacement

Next, cyclic peptides with some replaced amino acid were subjected tofollowing examination in order to confirm their effect. The methods forpreparing the above cyclic peptide and confirming amino acid sequence ofthe prepared peptide were same as the method for preparing the cyclicpeptide as described above and Mass spectroscopy method.

9.1 Confirming Effects on Scalp or Hair

The obtained cyclic peptide was prepared in purified water at 3, 15, 30,100 or 500 μg/ml and applied to the affected site. Each of formulationsA to E in the table indicates the concentration of cyclic peptide at 3,15, 30, 100 or 500 μg/ml, respectively. The absence of the effect uponthe application of purified water confirmed the effect was not a placeboeffect.

TABLE 29 SEQ ID NO (amino acid For- sequence) of mu- the cyclic la-Diagnostic impression of hair peptide used ID Sex Age Cases tionTreatment and scalp after application SEQ ID NO 16:   S1 male 50'smultiple alopecia C once a day After 1 day, fluffy hairs (CFVRKMDRISSareata started to grow. After 3 days, SSGLGC)obviously Hair started to grow, restoration and elongation wereobserved. Hair gained elasticity and resilience, thickened. Hairroot regeneration confirmed.   S2 fe- 40's seborrheic derma- Conce a day Iches were remitted immediately male titis male AGAafter applicaation. After 3 minutes, seborrhea and erythemaremitted. After 1 day, dandruff, redness and seborrhea of scalpimproved, falling hairs reduced. After 7 days, hairs stimulatedto grow, restored hair and hair growth confirmed.   S3 fe- 50'sfemale AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume.   S4 fe- 50's hair thinning Conce a day After 5 days, hair stimulation male after anticancerand growth confirmed. Hair became agent therapy dense and thick.   S5fe- 20's multiple alopecia C once a day Iches were improved immediatelymale areata after application. After 1 day,fluffy hairs started to grow. After 3 days, obviously Hairgrowth stimulated and restored elongation of hair observed, hairgained elasticity and resilience, thickened. Hair root regenerationobserved.   S6 fe- 60's healthy skin  C once a dayAfter 1 day, hair became fuller male (scalp) with increased volume. Hairgained elasticity and resilience. After 7 days, grown hairs werepredominantly black over white hairs. White hairs turned black.SEQ ID NO 17:   S7 male 40's multiple alopecia B once a dayAfter 3 days, remarkable terminal (CFGQKMDRISSS areatahair growth was observed. SGLGC)   S8 fe- 20's multiple alopecia Bonce a day After 5 days, newly born hair male areataroots and grown hairs were observed.   S9 male 30's male AGA Bonce a day After 2 or 3 days, hair gainedelasticity and resilience. After 7 days, hair gained volume,restoration was confirmed.  S10 fe- 30's female alopecia C once a dayHair gained elasticity and resi- male lience, hair volume was increasedon the night of first applica- tion. After 5 days, appearance ofscalp through hair were improved. The effect lasted for 1 weekafter stopping application. SEQ ID NO 18:  S11 male 60's male alopecia Bonce a day After 1 week, hair gained thick- (CFGHKMDRISSSness and elasticity, hair density SGLGC) was increased.  S12 male 70'shealthy skin  C once a day After 1 day, hair became fuller (scalp)with increased volume. Hair gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black.  S13 fe- 40's alopecia areata Bonce a day After 5 days, hair started to malegrow, elongation and growth of hair were observed.  S14 fe- 60'salopecia areata B once a day After 5 days, hair started to malegrow, hair density was increased. SEQ ID NO 19:  S15 fe- 20'smultiple alopecia B once a day After 5 days, hair started to(CFGRRMDRISSS male areata grow, elongation and restoration SGLGC)of hair were observed.  S16 fe- 50's hair thinning C once a dayAfter 2 days, hair gained elasti- male after anticancercity and resilience volume of agent therapy hair increased  S17 male60's male AGA C once a day After 1 day, hair gained elasti-city, resilience and increased volume. Hair started to grow,restoration and elongation were observed  S18 fe- 40's female alopecia Donce a day After 3 days, scalp shown through malehair due to thinning got ob- secure. Reduced falling hairs.  S19 fe-40's scalp seborrheic C once a day After 3 days, seborrhea and maledermatitis erythema were improved, so was dandruff. SEQ ID NO 20:  S20male 50's male AGA C once a day After 1 day, hair gained elasti-(CFGRKLDRISSS city, resilience and increased SGLGC) volume.  S21 male 40's multiple alopecia C once a day After 3 days, hair density wasareata increased. Hair started to grow and grew. SEQ ID NO 21:  S22 male40's multiple alopecia C once a day After 3 days, hair started to(CFGRKIDRISSS areata grow at the site of alopecia SGLGC)areata and grew.  S23 male 40's male alopecia C 1 applica-After 3 days, hair started to tion/day on gain resilience, felt thick. parietal After 7 days, parietal thinning male thin-were improved and hair volume ning area started to increase.  S24 fe-40's female AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume. Hair started to grow,restoration and elongation were observed.  S25 fe- 30'smultiple alopecia C once a day After 1 day, hair started to  male areatagrow. After 2 weeks, obvious lots of hairs started to grow, restor-ation, elongation and growth were observed.  S26 fe- 10'smultiple ophiasis C once a day After 2 days, stimulated hair malegrowth was confirmed. After 7 days, obvious stimulated hairgrowth was observed, elongated and grown hair was also observed.SEQ ID NO 22:  S27 male 40's male AGA B 1 applica-After 2 or 3 days, hair gained (CFGRKMDRVSSS tion/day onelasticity and resilience. After SGLGC) forehead7 days, hair gained volume, thinning restoration was confirmed. area S28 male 20's multiple alopecia C once a dayAfter 3 days, hair started grow areata and grew.  S29 fe- 60'sfemale AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume.  S30 fe- 70's female alopecia Conce a day After 7 days, increased hair malevolume. Appearance of scalp shown through hair got obsecure.  S31 fe-20's ophiasis B once a day After 5 days, stimulated hair maleconfirmed along intractable hair- line, and elongation of hair observed. S32 fe- 30's multiple alopecia C once a dayAfter 1 day, hair started to  male areatagrow. After 6 weeks, obvious stim- ulated hair growth, elongationand growth of hair were observed SEQ ID NO 23:  S33 male 30'salopecia areata C once a day After 3 days, stimulated and (CFGRKMDRIGSSuniversalis elongated hairs were observed SGLGC)  S34 fe- 60'shair thinning C once a day After 1 day, hair gained elasti- maleafter anticancer city and resilience. agent therapy  S35 fe- 50'sfemale AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume. Hair started to grow,restoration and elongation were observed.  S36 fe- 40's female alopeciaB 1 applica- After 5 days, hair gained elasti- male tion/day oncity, resilience and increased parietal volume. Appearance of scalpand fore- shown through hair got obsecure. head thin- ning area  S37male   60's multiple alopecia C once a dayAfter 1 day, stimulated hair was areata observed. After 3 days, applica-tion was stoped, but stimulation and elongation of hair contined.White hairs turned black again, black hairs started to grow.  S38 fe-40's female alopecia B once a day After 7 days, hair gained volume, maleseborrheic derma- appearance of scalp shown through titishair got obsecure. Seborrhea of scalp was improved.  S39 fe- 10'sophiasis B once a day After 4 days, regardless of high- malely intractable ophiasis, stimu- lated soft hairs were observed.  SEQ ID NO 24:  S40 fe- 10's alopecia areata A once a dayAfter 7 days, stimulated and  (CFGRKMDRISAS malegrown soft hairs were observed. SGLGC)  S41 fe- 50's female AGA Conce a day After 1 day, hair gained elasti- malecity, resilience and increased volume.  S42 male 30's multiple alopeciaB once a day After 4 days, stimulation and areataelongation of hair observed.  S43 fe- 30's multiple alopecia Conce a day After 1 day, hair started to  male areatagrow. A number of stimulated, elongated and grown hairs wereobserved in 3 weeks. SEQ ID NO 25:  S44 male 60's multiple alopecia Bonce a day After 5 days, hairs started to (CFGRKMDRISSQ areatagrow and grew. SGLGC)  S45 fe- 50's male AGA C once a dayAfter 1 day, hair gained elasti- male city, resilience and increasedvolume. Hair started to grow, restoration and elongation were observed. S46 fe- 60's multiple alopecia B once a dayAfter 5 days, hair root regenera- male areatation and hair growth stimulation were observed. Application con-tinued and a remarkable restora- tion, elongation and growth ofhair were observed. Hair elonga- tion rate was fast.  S47 fe- 40'sfemale alopecia C once a day After 1 day, hair gained elasti- malecity and resilience. Application continued for 3 days and stopped,but hair kept elasticity and resilience. The effect lasted for 1 week.SEQ ID NO 26:  S48 fe- 30's female alopecia C once a dayAfter 3 days, appearance of scalp (CFGRKMDRISSV maleshown through hair got obsecure, SGLGC) hair gained elasticity, resili-ence and increased volume. Hairs were restored.  S49 male 30'smale alopecia B once a day After 5 days, volume of hairincreased. Hair gained elasticity and resilience.  S50 fe- 20'sfemale AGA C once a day Volume of hair increased and malescalp does not show through after 3 days. The effect went on  S51 fe-60's female AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume. Hair started to grow,restoration and elongation were observed.  S52 male 20'smultiple alopecia C once a day After 3 days, stimulated hair was areataobserved After 7 days, further stimulation and growth of hair were con-firmed.  S53 fe- 30's multiple alopecia C once a dayAfter 1 day, hair started to male areata grow. Terminal hairs started togrow, and restoration, elongation and growth of hair were observedin 2 weeks. SEQ ID NO 27:  S54 fe- 50's hair thinning B once a dayAfter 5 days, hair got thick and (CFGRKMDRISSL male after anticancerdense. SGLGC) agent therapy  S55 male 50's male AGA C once a dayAfter 1 day, hair gained elasti- city, resilience and increasedvolume. Hair started to grow, restoration and elongation were observed. S56 male 20's multiple alopecia E once a dayAfter 3 days, hair started to areata grow After 7 days, stimulation andgrowth of hair were confirmed. SEQ ID NO 28:  S57 fe- 40's female AGA Conce a day After 1 day, hair gained elasti- (CFGRKMDRISSI malecity, resilience and increased SGLGC) volume.  S58 male 20'smultiple alopecia C once a day After 1 day, hair falling was re- areataduced and hair started to grow. After 7 days, remarkable stimula-tion, elongation and growth of hair were confirmed.  S59 male 20'smultiple alopecia C once a day After 1 day, hair started to grow areataand falling hairs reduced. After 4 days, application was stoped,but stimulation, elongation and restoration of hair persisted.  S60 fe-50's healthy skin  C once a day After 1 day, hair became fuller male(scalp) with increased volume. Hair gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black.  S61 fe- 40's multiple alopecia Conce a day After 3 days, hairs started to male areata grow and grew. S62 fe- 40's alopecia areata B once a day After 5 days, stimulated hairmale growth was confirmed. After 2 weeks, further growth of terminalhair, elongation and growth of hair were confirmed all overalopecia site. SEQ ID NO 29:  S63 fe- 40's female alopecia C once a dayAfter 4 days, hair gained elasti- (CFGRKMDRISSM malecity, resilience and increased SGLGC) volume.  S64 male 40'shealthy skin  C once a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience.  S65 male 40's male AGAC once a day Hair gained resilience and volumeafter 3 days. Application con- tinued for 1 week, and the effectpersisted for 1.5 months there- after.  S66 male 30's male alopecia C2 applica- After 3 days, hair gained elasti- tion/daycity, resilience and increased for 3 daysvolume at thinning area on fore- head. After a few days, hairgrowth stimulation and hair growth were confirmed.  S67 fe- 70'sfemale alopecia C once a day After 2 days, hair gained elasti- malecity and resilience. After 5 days, volume of hair increased.It was confirmed that the stimu- lated hairs are predominantlyblack over white hair. SEQ ID NO 30:  S68 fe- 20's alopecia areata Bonce a day After 7 days, stimulated hair (CFGRKMDRISSS male ophiasisgrowth was confirmed. Even 2 VGLGC) weeks after stopping application,hair growth stimulation and re- storation persisted, and therewere little falling hairs.  S69 male 40's male AGA B once a dayAfter 2-3 days, hair gained elasticity and resilience. After7 days, hair gained volume, re- storation was confirmed.  S70 male 30'salopecia univer- B once a day After 3 days, hairs started to salisgrow and grew.  S71 fe- 30's multiple alopecia C once a dayAfter 1 day, stimulated hair male areata growth was confirmed.SEQ ID NO 31:  S72 fe- 20's alopecia areata C once a dayAfter 7 days, stimulated hair (CFGRKMDRISSS male ophiasisgrowth was confirmed. Even 2 SRLGC) weeks after stopping application,hair growth stimulation and re- storation persisted, and fallinghairs significantly decreased  S73 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (scalp) creased. Hair gained elasticityand resilience.  S74 male 30's alopecia areata C 2 applica-After 3 days, stimulation and universalis tion/dayelongation of hair were con- for 3 days firmed.  S75 fe- 60'smultiple alopecia B once a day After 5 days, hairs stimulated to malegrow, restored hair and hair growth were confirmed. It wasconfirmed that hair elongation rate is fast. SEQ ID NO 32:  S76 male30's male alopecia B once a day After 7 days, hair gained elasti-(CFGRKMDRISSS city. Hair started to grow, and SGMGC)volume of hair on parietal fore- head was increased.  S77 fe- 60'sfemale AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume. Hair started to grow,restoration and elongation were observed.  S78 male 40'salopecia univer- C once a day After 3 days, hair stimulation  salisand growth were confirmed  S79 fe- 50's hair thinning C once a dayAfter 2 days, hair gained elasti- male after anticancercity, resilience and increased agent therapyvolume. Appearance of scalp shown through hair got obsecure. Hairincrease was confirmed. SEQ ID NO 33:  S80 fe- 50's multiple alopecia Bonce a day After 7 days, stimulated hair (CFGRKMDRISSS male areatagrowth was confirmed. SGIGC)  S81 male 60's healthy skin C once a dayAfter 1 day, hair became fuller (scalp with increased volume. Hairgained elasticity and resilience. After 7 days, grown hairs werepredominantly black over white hairs. White hairs turned black.  S82 fe-40's multiple alopecia B once a day After 5 days, hair root regenera-male areata tion and hair growth stimulation were confirmed.   S83 male60's alopecia areata C once a day After 1 day, apparent hair growthstimulation and restoration were confirmed. After 4 days, hairelongation were confirmed. SEQ ID NO 34:  S84 fe- 50's hair thinning Bonce a day After 5 days, thin, fuzzy hairs (CFGRKMDRISSS maleafter anticancer along hairline got thicker and SGVGC) agent therapydenser.  S85 male 50's male AGA C once a dayAfter 1 day, hair gained elasti- city, resilience and increasedvolume. Hair started to grow, restoration and elongation were observed. S86 male 40's male AGA C once a day Hair gained resilience and volumeafter 3 days. Application con- tinued for 1 week, and the effectpersisted for 1.5 months there- after.  S87 male 30's male alopecia Conce a day After 3 days, hair gained resili-ence and volume of hair increased in parietal thinning area  S88 male20's multiple alopecia C once a day After 1 day, hairs started to areatagrow and grew. SEQ ID NO 35:  S89 fe- 40's hair thinning B once a dayAfter 5 days, hair gained elasti- (CFGRKMDRISSS male after anticancercity, resilience, thickness, and SGAGC) agent therapy increased volume. S90 male 20's multiple alopecia C once a dayAfter 3 days, stimulated hair  areata growth was confirmed.  S91 fe-50's female AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume. Hair started to grow,restoration and elongation were observed.  S92 fe- 60'smultiple alopecia B once a day After 5 days, hair root regenera- maleareata tion and hair growth stimulation were observed. Then, remarkablerestoration, elongation and  growth of hair were observed.  S93 fe- 30'smultiple alopecia C once a day After 1 day, stimulated hair was maleareata observed. After 2 weeks, there is remarkable elongation of hairmarginal area around large de- calvant spot, and new hair start-ed to grow in center part. SEQ ID NO 36:  S94 male 30's alopecia univer-C once a day After 4 days, hair stimulation (CFGRKMDRISSS salisand growth were confirmed. SGLSC)  S95 male 40's healthy skin Conce a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience.  S96 male 50'sseborrheic derma- C once a day After 3 days, seborrhea and  titiserythema were improved.  S97 male 50's male alopecia B once a dayAfter 5 days, hair gained elasti- city, resilience and increased volume.SEQ ID NO 37:  S98 fe- 40's hair thinning B once a dayAfter 5 days, hair gained elasti- (CFGRKMDRISSS male after anticancercity, resilience, thickness and SGLAC) agent therapy increased volume. S99 male 40's male AGA B 1 applica- After 2 or 3 days, hair gainedtion/day on elasticity and resilience. After forehead7 days, hair gained volume, re- thinning  storation was confirmed. areaS100 male 20's multiple alopecia C once a dayAfter 5 days, stimulated hair areata growth was confirmed. S101 fe- 20'salopecia areata A once a day After 10 days, hair growth stimu- malelation elongation were confirmed. SEQ ID NO 38: S102 fe- 50's female AGAC once a day After 1 day, hair gained elasti- (CFARKMDRISSS malecity, resilience and increased SGLGC) volume. Hair started to grow,restoration and elongation were observed. S103 fe- 50's dandruff, itchesC once a day Iches stopped in 3 minutes,  maledandruff was reduced, redness in scalp was significantly improved. S104fe- 10's eclopic multple C once a day After 3 days, stimulated hair malealopecia growth was confirmed in eyebrows. SEQ ID NO 39: S105 male 10'salopecia univer- C once a day After 3 days, stimulated hair(CFSRKMDRISSS salis growth was confirmed. SGLGC) S106 fe- 50'sdandruff, itches C once a day Iches stopped in 1 minute, dan- maledruff was reduced, redness in scalp was significantly improved. S107 fe-60's healthy skin C once a day After 1 day, hair became fuller male(scalp) with increased volume. Hair gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black. S108 fe- 60's multiple alopecia Conce a day After 3 days, stimulated hair male areatagrowth was confirmed. More black hairs started to grow than whitehairs, and hair elongation rate was fast. SEQ ID NO 40: S109 male 60'smale AGA C once a day After 1 day, hair gained elasti- (CFTRKMDRISSScity, resilience and increased SGLGC) volume. Hair started to grow,restoration and elongation were observed. S110 male 20'smultiple alopecia C once a day After 4 days, stimulated hair areatagrowth was confirmed C once a day SEQ ID NO 41: S111 fe- 50's female AGAC once a day After 1 day, hair gained elasti- (CFGRKMDRISST malecity, resilience and increased SGLGC) volume. Hair started to grow,retoration and elongation were observed. S112 male 20'smultiple alopecia C once a day After 4 days, stimulated hair areatagrowth was confirmed. SEQ ID NO 42: S113 male 30's multiple alopecia Conce a day After 3 days, stimulated hair (CFGRKMDRISSA areatagrowth was confirmed. SGLGC) S114 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (scalp) creased. Hair gained elasticityand resilience. S115 fe- 60's multiple alopecia C once a dayAfter 3 days, stimulated hair male areatagrowth was confirmed. Black hairs started to grow and hair elonga-tion was fast SEQ ID NO 43: S116 male 40's male AGA B 1 applica-After 2-3 days, hair gained (CFGRKMDRISSS tion/day onelasticity and resilience. After TGLGC) forehead7 days, hair gained volume, re- thinning storation was confirmed areaS117 male 20's multiple alopecia D once a dayAfter 3 days, stimulated hair areata growth was confirmed. SEQ ID NO 44:S118 fe- 60's female AGA C once a day After 1 day, hair gained elasti-(CFGRKMDRISSS male city, resilience and increased AGLGC)volume. Hair started to grow, restoration and elongation were observedS119 fe- 20's multiple alopecia C once a dayAfter 3 days, stimulated hair male areata growth was confirmed.SEQ ID NO 45: S120 male 40's male AGA C once a dayAfter 1 day, hair gained elasti- (CFSRRMDRISSScity, resilience and increased SGLGC) volume. Hair started to grow,restoration and elongation were observed. S121 fe- 40's healthy skin Conce a day After 1 day, hair volume was in- male (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 46: S122 fe-60's female AGA C once a day After 1 day, hair gained elasti-(CFSRKMDRISST male city, resilience and increased SGLGC)volume. Hair started to grow, restoration and elongation were observed.S123 fe- 60's healthy skin C once a day After 1 day, hair became fullermale (scalp) with increased volume. Hairgained elasticity and resilience. After 7 days, grown hairs werepredominantly black over white hairs. White hairs turned black.SEQ ID NO 47: S124 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (CFSRKMDRISSS (scalp)creased. Hair gained elasticity TGLGC) and resilience. S125 fe- 50'sfemale AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume. Hair started to grow, re-storation and elongation were observed. SEQ ID NO 48: S126 fe- 40'smultiple alopecia B once a day After 5 days, hair root regenera-(CFSRKMDRISSS male areata tion and hair growth stimulation SGIGC)were confirmed S127 fe- 60's healthy skin C once a dayAfter 1 day, hair became fuller male (scalp) with increased volume. Hairgained elasticity and resilience. After 7 days, grown hairs werepredominantly black over white hairs. White hairs turned black S128 fe-50's female AGA C once a day After 1 day, hair gained elasti- malecity, resilience and increased volume. Hair started to grow,restoration and elongation were observed. SEQ ID NO 49: S129 fe- 60'sfemale AGA C once a day After 1 day, hair gained elast- (CFSRKMDRISSSmale city, resilience and increased SGLAC) volume. S130 fe- 60'salopecia areata B once a day After 5 days, hair started to malegrow and hair density was in- creased S131 fe- 40's healthy skin Conce a day After 1 day, hair volume was in- male (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 50: S132 male50's male AGA C once a day After 1 day, hair gained elasti-(CFGRRMDRISST city, resilience and increased SGLGC)volume. Hair started to grow, re- storation and elongation wereobserved. S133 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (scalp) creased. Hair gained elasticityand resilience. SEQ ID NO 51: S134 male 40's male AGA B 1 applica-After 2 or 3 days, hair gained (CFGRRMDRISSS tion/day onelasticity and resilience. After TGLGC) forehead7 days, hair gained volume, re- thinning storation was confirmed. areaS135 male 50's healthy skin C once a day After 1 day, hair became fuller(scalp) with increased volume. Hair gained elasticity and resilience.SEQ ID NO 52: S136 male 60's male AGA C once a dayAfter 1 day, hair gained elasti- (CFGRRMDRISSScity, resilience and increased SGIGC) volume. Hair started to grow, re-storation and elongation were observed. S137 fe- 40's multiple alopeciaB once a day After 5 days, hair root regenera- male areatation and hair growth stimulation were confirmed. S138 fe- 60'shealthy skin C once a day After 1 day, hair became fuller male (scalp)with increased volume. Hair gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black. SEQ ID NO 53: S139 fe- 40's female AGAC once a day After 1 day, hair gained elasti- (CFGRRMDRISSS malecity, resilience and increased SGLAC) volume. S140 male 40'shealthy skin C once a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 54: S141 male40's male AGA B 1 applica- After 2 or 3 days, hair gained (CFGRKMDRISSTtion/day on elasticity and resilience. After SGIGC) forehead7 days, hair gained volume, re- thinning storation was confirmed. areaS142 fe- 50's healthy skin C once a day After 1 day, hair became fullermale (scalp) with increased volume. Hairgained elasticity and resilience. After 7 days, grown hairs werepredominantly black over white hairs. White hairs turned black.SEQ ID NO 55: S143 male 50's male AGA C once a dayAfter 1 day, hair gained elasti- (CFGRKMDRISSTcity, resilience and increased SGLAC) volume. Hair started to grow, re-storation and elongation were observed. S144 fe- 60's healthy skin Conce a day After 1 day, hair became fuller male (scalp)with increased volume. Hair  gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black. SEQ ID NO 56: S145 male 40's male AGA Conce a day After 1 day, hair gained elasti- (CFGRKMDRISSScity, resilience and increased TGIGC) volume. Hair started to grow, re-storation and elongation were observed. S146 male 40's healthy skin Conce a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 57: S147 male50's male AGA C once a day After 1 day, hair gained elasti-(CFGRKMDRISSS city, resilience and increased TGLAC)volume. Hair started to grow, re- storation and elongation wereobserved. S148 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (scalp) creased. Hair gained elasticityand resilience SEQ ID NO 58: S149 fe- 40's female AGA C once a dayAfter 1 day, hair gained elasti- (CFGRKMDRISSS malecity, resilience and increased SGIAC) volume. S150 fe- 40's healthy skinC once a day After 1 day, hair volume was in- male (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 59: S151 male50's male AGA C once a day After 1 day, hair gained elasti-(CFSRRMDRISST city resilience and increased SGLGC)volume. Hair started to grow, re- storation and elongation wereobserved. S152 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (scalp) creased. Hair gained elasticityand resilience. SEQ ID NO 60: S153 male 60's male AGA C once a dayAfter 1 day, hair gained elasti- (CFSRRMDRISSScity, resilience and increased TGLGC) volume. Hair started to grow, re-storation and elongation were observed. S154 male 40's healthy skin Conce a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 61: S155 fe-60's female AGA C once a day After 1 day, hair gained elasti-(CFSRRMDRISSS male city, resilience and increased SGIGC)volume. Hair started to grow, re- storation and elongation wereobserved. S156 male 60's healthy skin C once a dayAfter 1 day, hair became fuller (scalp) with increased volume. Hairgained elasticity and resilience. After 7 days, grown hairs werepredominantly black over white hairs. White hairs turned black.SEQ ID NO 62: S157 male 50's male AGA C once a dayAfter 1 day, hair gained elasti- (CFSRRMDRISSScity, resilience and increased SGLAC) volume. Hair started to grow, re-storation and elongation were observed. S158 male 40's healthy skin Conce a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 63: S159 male60's male AGA B 1 applica- After 2 or 3 days, hair gained (CFSRKMDRISSTtion/day  elasticity and resilience. After SGIGC) on forehead7 days, hair gained volume, re- thinning storation was confirmed. areaS160 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (scalp) creased. Hair gained elasticityand resilience. SEQ ID NO 64: S161 male 60's healthy skin C once a dayAfter 1 day, hair became fuller (CFSRKMDRISSS (scalp)with increased volume. Hair TGIGC) gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black. S162 fe- 50's female AGA C once a dayAfter 1 day, hair gained elasti- male city, resilience and increasedvolume. Hair started to grow, re- storation and elongation were observedSEQ ID NO 65: S163 male 40's male AGA B 1 applica-After 2 or 3 days, hair gained (CFSRKMDRISSS tion/day onelasticity and resilience. After TGLAC) forehead7 days, hair gained volume, re- thinning storation was confirmed. areaS164 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (scalp) creased. Hair gained elasticityand resilience. SEQ ID NO 66: S165  male 60's male AGA C once a dayAfter 1 day, hair gained elasti- (CFSRKMDRISSScity, resilience and increased SGIAC) volume. Hair started to grow, re-storation and elongation were observed. S166 male 40's healthy skin Conce a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 67: S167 male40's male AGA B 1 applica- After 2-3 days, hair gained  (CFGRRMDRISSTtion/day on elasticity and resilience. After SGIGC) forehead7 days, hair gained volume, re- thinning storation was confirmed. areaS168 fe- 60's healthy skin C once a day After 1 day, hair became fullermale (scalp) with increased volume. Hairgained elasticity and resilience. After 7 days, grown hairs werepredominantly black over white hairs. White hairs turned black.SEQ ID NO 68: S169 fe- 40's female AGA C once a dayAfter 1 day, hair gained elasti- (CFGRRMDRISST malecity, resilience and increased SGLAC) volume. S170 male 40'shealthy skin C once a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 69: S171 male60's male AGA B 1 applica- After 2 or 3 days, hair gained (CFGRRMDRISSStion/day on elasticity and resilience. After TGIGC) forehead7 days, hair gained volume, re- thinning storation was confirmed. areaS172 male 30's multiple alopecia C once a dayAfter 1 day, hairs started to areata grow and grew. S173 male 60'shealthy skin C once a day After 1 day, hair became fuller (scalp)with increased volume. Hair gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black. SEQ ID NO 70: S174 fe- 50's female AGAC once a day After 1 day, hair gained elasti- (CFGRRMDRISSS malecity, resilience and increased TGLAC) volume. S175 male 40'shealthy skin C once a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 71: S176 male60's male AGA C once a day After 1 day, hair gained elasti-(CFSRRMDRISST city, resilience and increased SGIGC)volume. Hair started to grow, re- storation and elongation wereobserved. S177 male 20's multiple alopecia C once a dayAfter 1 day, hairs started to areata grow and grew. S178 male 60'shealthy skin C once a day After 1 day, hair became fuller (scalp)with increased volume. Hair gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black. SEQ ID NO 72: S179 male 40's male AGA B1 applica- After 2 or 3 days, hair gained (CFSRRMDRISST tion/day onelasticity and resilience. After SGLAC) forehead7 days, hair gained volume, re- thinning storation was confirmed. areaS180 male 40's healthy skin C once a dayAfter 1 day, hair volume was in- (scalp) creased. Hair gained elasticityand resilience. SEQ ID NO 73: S181 fe- 40's female AGA C once a dayAfter 1 day, hair gained elasti- (CFSRRMDRISSS malecity, resilience and increased TGIGC) volume. S182 fe- 40'salopecia areata B once a day After 5 days, hair started to malegrow and hair density was in- creased. S183 fe- 60's healthy skin Conce a day After 1 day, hair became fuller male (scalp)with increased volume. Hair gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black. SEQ ID NO 74: S184 male 60's male AGA Conce a day After 1 day, hair gained elasti- (CFSRRMDRISSScity, resilience and increased TGLAC) volume. Hair started to grow, re-storation and elongation were observed. S185 male 40's healthy skin Conce a day After 1 day, hair volume was in- (scalp)creased. Hair gained elasticity and resilience. SEQ ID NO 75: S186 male40's male AGA B 1 applica- After 2 or 3 days, hair gained (CFSRRMDRISSStion/day on elasticity and resilience. After SGIAC) forehead7 days, hair gained volume, re- thinning storation was confirmed. areaS187 fe- 40's multiple alopecia B once a dayAfter 5 days, hair root regenera- male areatation and hair growth stimulation were confirmed. S188 fe- 50'shealthy skin C once a day After 1 day, hair became fuller male (scalp)with increased volume. Hair gained elasticity and resilience.After 7 days, grown hairs were predominantly black over whitehairs. White hairs turned black. In SEQ ID NOS 16-75. 1st Cys and 17thCys from left form a disulfide bond.

The result described above demonstrated that the cyclic peptide of theinvention exhibits a similar effect as the cyclic peptide expressed byFormula I-a even if some amino acids of the cyclic peptide have beenreplaced.

Specifically, it has effects of preventing hair loss at the appliedsite, stimulating hair growth or promoting hair growth, giving hairresilience and elasticity, increasing volume of hair and dramaticallydecreasing falling hairs when it was applied to the site of alopecia orhair growth stimulation, etc. It also has effects of nourishing hair,promoting hair growth stimulation, improving/preventing thinning of hairat the applied site. In this case, the stimulated hair tends to become aterminal (non-white) hair. Moreover, these effect exhibit significantlyfaster as compared to other active agents conventionally used inalopecia therapeutics such as BNP such that stimulated hair growth canbe confirmed one day after only one application even in the case of anintractable alopecia areata multilocularis or ophiasis. In addition,resulting effects persists remarkably even after terminating applicationand the stimulated hair keep growing. In the case of AGA or healthysubject it exert immediate effect of providing hair with resilience andelasticity on the following day, increasing the volume, and dramaticallydecreasing falling hairs, which effects last even after terminatingapplication. It also has an improving and preventing effect ondermatitis. Therefore, it can improve or prevent skin inflammationassociated with alopecia. Such effect is advantageous in cases wherealopecia has been exacerbated due to skin condition of the applied site(e.g., scalp). Moreover, the external preparation of the presentinvention exerts a moisturizing and skin texture-improving effects atthe applied site when being applied to skin as described above. It canremove and suppress dandruff and itching, while giving moisture to hairand scalp, improving and preventing dryness and keeping hair and scalphealthy. It also can improve seborrhea. On the other hand, when it isused as an alopecia therapeutic/prophylactic, it can be used for thepurpose of treating or preventing one or more of applicable alopeciaincluding, for example, those described below, without beingparticularly limited thereto.

(Acquired Alopecia)

(i) Alopecia without accompanying scarring or skin lesion (alopeciaareata, male pattern alopecia, seborrheic alopecia, alopecia pityroides,female pattern alopecia, gestational alopecia, malignant alopecia,senile alopecia, alopecia totalis, alopecia areata multilocularis,ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopeciaand radiation exposure-induced alopecia, traumatic/mechanical alopecia,malnutrition/metabolic disorder associated alopecia, endocrinedysfunction associated alopecia and telogen effluvium (post partumalopecia, alopecia after high fever)).(ii) Alopecia observed in skin lesion or pathologic skin(infection-induced alopecia, tumor-induced alopecia,inflammation-induced alopecia).(iii) Scarring alopecia (skin infection-induced alopecia, alopeciainduced by infiltration of inflammatory cells).

(Congenital Alopecia)

Diffuse alopecia, congenital atrichia, alopecia in hereditary syndromes,localized alopecia, phakomatosis, aplasia cutis, congenital alopeciatriangularis.

It exerts an excellent effect especially on acquired alopecia,preferably on alopecia without accompanying scarring or skin lesion,more preferably on alopecia areata, male pattern alopecia, seborrheicalopecia, alopecia pityroides, female pattern alopecia, gestationalalopecia, malignant alopecia, senile alopecia, alopecia totalis,alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancerchemotherapy-induced alopecia and radiation exposure-induced alopecia,traumatic/mechanical alopecia, malnutrition/metabolic disorderassociated alopecia, endocrine dysfunction associated alopecia andtelogen effluvium. Therefore, the external preparation of the presentinvention can be used for the purpose of treating or preventing at leastone or more alopecia selected from the above-mentioned group.

One simple application onto aforementioned affected site at 3 μg/ml to500 μg/ml improved or eliminated itch, dandruff, scalp redness andinflammation immediately thereafter (immediate effect). A singleapplication stimulated hair growth, grew hair, provided hair withresilience, elasticity and increased volume, and dramatically decreasedfalling hairs, and these effects started on the day after theapplication and persisted for one week or more (long-lasting effect). Ifapplication was carried on for about one week, these effects are furtherimproved, and even after terminating application, the effects lastedfrom 1 to 2 weeks such that stimulation of hair growth and restorationof hair continued and there were little or no falling hairs.

9.2 Confirming Effects on Skin

The obtained cyclic peptide was prepared in purified water at 3, 15, 30,100 or 500 μg/ml and applied to affected site. Each of formulations A toE in the table indicates the concentration of the cyclic peptide at 3,15, 30, 100 or 500 μg/ml, respectively. The absence of the effect uponthe application of purified water without the peptide confirmed theeffect was not a placebo effect.

TABLE 30 Amino acid sequence of cyclic peptide  DiagnosticDiagnostic impression on skin  Itches after  mutant used ID Sex Ageimpression Formulation Treatment after application application 1SEQ ID NO 16: Q1  female 30′s eczema B once a dayAfter 3 minutes, red papule was reduced  Itches were remitted (CFVRKMDRISSSSGLGC) and remitted. immediately after  application. Q2 male 20′s atopic  B once a day After 2 minutes, erythema, edema and Itches were eliminated dermatitis infiltration was remitted.immediately after  application. Q3  male 80′s plaque  C once a day, 2After 3 minutes, crusts, scales, erythema psoriasis daysand infiltration were remitted. Q4  male 40′s plaque  C once a day,After 10 minutes, scales, erythema and  psoriasisinfiltration were remitted. Q5  female 30′s eczema C once a dayAfter 3 minutes, erythema was remitted. Q6  male 20′s atopic  Bonce a day. 7 Erythema, infiltration and papule were Itches were remitted  dermatitis days remitted. immediately after application. Q7  female 60′s eczema C once a dayAfter 2 minutes, erythema, infiltration  and papule were remitted.After 20 minutes, they were amaliorated. Q8  female 20′s atopic  Conce a day After 2 minutes, erythema and  Itches were eliminateddermatitis infiltration were remitted. in 1 minute Q9  male 10′s atopic C once a day Redness vanished immediately after  Itches were extincted dermatitis application. in 1 minute. SEQ ID NO 17: Q10 male 40′s atopic C once a day After 3 minutes, erythema and  After 3 minutes, (CFGQKMDRISSSSGLGC) dermatitis infiltration were remitted.itches were remitted. Q11 male 10′s nummular C once a dayAfter 3 minutes, infiltration, erythema  eczemaexudate and scales were remitted. Q12 male 20′s eczema B once a dayAfter 2 minutes, scales and erythema  Iches were elimintaedwere remitted. With only 1 application, immediately after no recurrence for 7 days thereafter. application. 2 SEQ ID NO 18: Q13male 60′s milleria C once a day After 2 minutes, redness was remitted.(CFGQKMDRISSSSGLGC) Q14 female 40′s roughened A once a dayAfter 2 minutes, zits, dryness and  skinrawness on skin were reduced, skin was moisturized and it texture improved.  After 7 days, large wrinkles and nasolabial fold became less deep. SEQ ID NO 19: Q15 male 20′seczema, atopic C once a day After 2 minutes, erythema and scales were (CFGRRMDRISSSSGLGC) dermatitis remitted. After 20 minutes, skin got moisturized. After only 1 application, erythema vanishes and kept in a good  condition for 1 week. Q16 male20′s atopic  C once a day After 5 minutes, wound was apithelized, dermatitis erythema and papule were improved. Q17 female 40′s roughenedB once a day After 2 minutes, redness was remitted.  skinRoughness and skin texture were also  improved. Q18 female 30′sroughened B once a day After 2 minutes, was remitted. Roughness  skinand skin texture were also improved. Rawness got better, iches vanished  immediately after application, skin were moisturized and smooth. Q19 male 40′s plaque  C once a dayAfter 3 minutes, erythema, crusts scales  psoriasisand infiltration were remitted. Q20 male 40′s eczema C once a dayErythema was remitted immediately after  Itches were extinctedapplication. immediately after  application SEQ ID NO 20: Q21 female60′s eczema C once a day After 1 minute, red papule was reduced After 3 minutes,  (CFGRKLDRISSSSGLGC) and extincted. iches were remittedQ22 male 20′s atopic  B once a day After 2 minutes, erythema and scales Itches vanished  dermatitis were remitted. completely immediatelyafter application. Q23 female 40′s eczema C once a dayAfter 2 minutes, erythema, scales and  Rawness vanished papule were remitted. immediately after  Q24 female 30′s roughened Bonce a day After 3 minutes, zits, ruggedness, dry Iches application were skin, acneskin and redness were remitted, skin got  eliminated immediatelyfull and its texture improved. Acnes  after application.were reduced and redness was remitted. Q25 female 60′s wrinkles,  Bonce a day After 2 minutes, wrinkles became less  dry skindeep, dry skin was remitted. Q26 female 60′s eczema C once a dayAfter 3 minutes, erythema and scales  were remitted. SEQ ID NO 21: Q27male 20′s eczema, atopic C once a dayAfter 3 days, erythema, scales and  Iches were eliminated(CFGQKIDRISSSSGLGC) dermatitis papule were improved. Cracks were immediately after  epithelized, skin got moisturized and  application.improved. Q28 female 40′s eczema B once a dayAfter 2 minutes, infiltration, erythema,  After 1 minute, ichespapule and scratch wound were remitted. were eliminated. Q29 male 40′satopic  B once a day Immediately after application, erythemaAfter 3 minutes,  dermatitis and scales and remitted.iches were eliminated. 3 SEQ ID NO 22: Q30 female 30′s atopic  Conce a day After 3 minutes, 1 application, erythema  After 30 seconds, (CFGQKMDRISSSSGLGC) dermatitisand infiltration were remarkably remitted, Iches were eliminated.dry skin, scales and cracks were also  improved. Q31 female 70′s eczemaC once a day immediately after application, erythema  Facial rawness and papule remitted. vanished immediately  Q32 female 30′s atopic  Bonce a day, A remarkable improvement after 2 days,  after application.dermatitis 7 days and erythema was eliminated. Q33 female 40′s atopic  Conce a day, After 1 minute, erythema was remitted. Iches were eliminateddermatitis 2 days After 2 minutes, scratch wound was  immediately after epithelized. application After 3 minutes, skin texture was improved.Iches were eliminated With only 1-day applicatioin, erythema immediately after  was eliminated, skin texture was  applicationimproved with no dryness. Q34 male 20′s atopic  C once a dayAfter 2 minutes, erythema and scales were Iches were remitted dermatitis remitted. No recurrence for 7 days  immediately thereafter.after application Q35 female 50′s chronic eczema C once a dayAfter 2 applications, lichenification  Iches were eliminatederythema was remitted. No recurrence  immediately after for 3 weeks thereafter. application Q36 female 50′s roughened Conce a day Skin roughness was cured immediately  skinafter application. After 2 minutes, skin got moisturized. Its redness faded. Skin texture was finely improved. After 7 days, skin tone became brighter,spots  faded and flabiness was improved.Q37 female 30′s roughened B once a dayAfter 2 minutes, zits, redness was  Rawness vanished  skinremitted. After 1 day, skin became  immediately after normal tone and its texture was improved. application. SEQ ID NO 23: Q38female 40′s atopic  C once a day After 2 minutes, scratch wound was After 1 minute, acnes (CFGQKMDRISSSSGLGC) dermatitisepithelized and cured. Erythema was  were eliminated. improved. Q39 male20′s eczema C once a day After 2 minutes, erythema, dry skin and Itches were abeled  scales were improved and skin got immediately after  moisturized. application Q40 male 20′s atopic  Conce a day After 2 minutes, erythema, scales and  Itches were extincted dermatitis infiltration were remitted. With 1  immediately after application, no recurrence for 7 days. application. Q41 male 40′splaque  C once a day After 3 minutes, crustsscaleserythema  psoriasisand infiltration were remitted. Q42 female 30′s roughened B once a dayAfter 2 minutes, roughness, zits and  After 2 minutes,  skinredness were reduced, skin got moisturized itches were and its texture was improved. extincted. Q43 female 50′s rosacea, acne Conce a day After 1 minute, erythema, telangiectasia and acne were remitted. After 2 minutes, papule, infiltration, acne, dry skin and  redness was remitted. 4SEQ ID NO 24: Q44 male 10′s atopic  C once a dayAfter 3 minutes, erythema, scales and  (CFGQKMDRISASSGLGC) dermatitisinfiltration were remitted. Further improvement after 40 minutes. Scratch wound was epithelized. q45 male 20′s atopic  C once a dayAfter 3 minutes, erythema and  After 3 minutes,  dermatitisinfiltration were remitted. Iches were  Q46 male 20′s C once a dayAfter 3 days, cracks were epithelized.  eliminated.Erythema, scales and infiltration were  remitted. Q47 male 20′s atopic C once a day After 2 minutes, wound was epithelized. Iches were eliminated  dermatitis Erythema, scales and licherification immediately after  remitted. After 5 minutes, skin got  application.softened, erythema and infiltration were  improved. SEQ ID NO 25: Q48male 20′s C once a day After 2 minutes, erythema,scales and Iches were remitted  (CFGQKMDRISSQSGLGC)infiltration were remitted. Skin got  immediately after  moisturized.application. Q49 female 40′s roughened B once a dayAfter 3 minutes, deep wrinkles were less  Rawness was eliminated  skindeep, dry skin and redness were improved, immediately after skin got moisturized and full. By using  application.for 1 week, large wrinkles and nasolabialfold became less deep. Skin became  brighter. Spots faded.Flabiness was improved. SEQ ID NO 26: Q50 male 60′s plaque  C once a dayAfter 2 minutes, scales and infiltration  (CFGQKMDRISSSSGLGC) psoriasisremitted. Q51 male 30′s millaria C once a day After 2 minutes, remitted.Q52 male 20′s acne vulgaris C once a dayAfter 6 minutes, acne became dry and was  reduced in size. Q53 female60′s eczema C once a day After 3 minutes, erythema and infiltrationwere remitted. After 4 minutes, amelicrated. Q54 female 10′s atopic  Conce a day After 2 minutes, strong itches almost  dermatitisvanished. Erythema and lichenification were improved. Skin got softened. Q55 female 40′s roughened C once a dayAfter 2 minutes, zits, redness and  Rawness vanished  skinroughness were improved. Skin got  immediately after moisturized and its texture was  application.improved. Skin was improved. SEQ ID NO 27: Q56 female 50′s roughened Conce a day After 3 minutes, dry skin was improved,  (CFGQKMDRISSSSGLGC)skin, acne skin got moisturized and its texture was finely improved. Redness vanished.  Inframmation in acne was remitted, redness faded and dried. By using for 1  week, skin became brighter. Q57male 20′s atopic  C once a day After 3 minutes, erythema, infiltrationIches were eliminated dermatitis and lichenification was improved.immediately after  application. 5 SEQ ID NO 28: Q58 female 30′s eczema Conce a day After 1 minute, erythema and infiltration  After 1 minute, (CFGQKMDRISSISGLGC) were remitted. Iches were  Q59 female 20′s roughenedA once a day After 7-day continuous application, skin  eliminated. skinroughness vanished, wrinkles were less  deep, and skin became brighter.Q60 female 20′s roughened C once a dayAfter 2 minutes, scales were improved.  skinWith 1 application/day, roughness was  improved and skin could be kept moisturized. SEQ ID NO 29: Q61 female 30′s eczema C once a dayAfter 1 minute, erythema and  Iches were eliminated (CFGQKMDRISSMSGLGC)infiltration were remitted. immediately after  application.SEQ ID NO 30: Q62 male 20′s atopic  B once a day,After 3 days, erythema and infiltration (CFGQKMDRISSSVGLGC) dermatitis7 days were remitted. Q63 female 60′s eczema C once a dayAfter 1 minute, erythema and papule were  Iches were eliminatedremitted. After 3 days, there were  immediately after  almost cured.application. Q64 male 50′s dry skin C once a dayImmediately after application, skin got promptly moisturized and its texture was improved, zits, erythema and roughned  skin were improved. Q65 male 20′satopic  C once a day After 10 minutes, lichenification was Iches were eliminated dermatitis improved. Skin got softened, erythema immediately after  and scales were improved. application. Q66 female40′s roughened A once a day After 1 day with 1 application, skin  skinconditions of roughness, zits and itches were improved and skin texture was finely improved. The effect lasted for 7 days  thereafter. SEQ ID NO 31: Q67male 20′s atopic  C once a day After 2 minutes, erythema and papule (CFGQKMDRISSSSRLGC) dermatitis was remitted. SEQ ID NO 32: Q68 male 30′smillaria C once a day After 2 minutes, red papule was reduced (CFGQKMDRISSSSGMGC) in size, skin dryness was remitted. Q69 male 10′satopic  C once a day After 3 minutes, exudate stopped,  dermatitis,erythema and infiltration were improved. numeuler 6 SEQ ID NO 33: Q70female 30′s contact C 2 applicatioinAfter 3 minuts, erythema, infiltration  After 1 minute, iches(CFGQKMDRISSSSGIGC) dermatits and eczema were remitted. were eliminated.Q71 female 30′s atopic  E once a dayImmediately after application, erythema  Iches were eliminateddermatitis and scales were improved. immediately after  Q72 female 70′splaque  C 2 application After 5 minutes. erythema, scales  application.psoriasis and infiltration were remitted. Q73 female 40′s roughened Bonce a day In 2 minutes after application, skin  skinwas improved. Skin texture was finely  improved. Skin got softened.SEQ ID NO 34: Q74 female 30′s eczema C 2 applicatioinAfter 3 minutes, erythema, infiltration  After 1 minute, Iches(CFGQKMDRISSSSGVGC) and edema remitted. were eliminated. Q75 male 20′seczema, acne C 2 applicatioin After 5 minutes, acne was dried and vulgaris reduced in size. Erythema and scales  were remitted. Q76 female70′s plaque  C 2 applicatioin After 5 minutes, erythema, scales and psoriasis infiltration were remitted. Q77 female 40′s roughened Conce a day After 2 minutes, skin texture was  skinimproved, skin got hull, and redness  and dryness were improved. Q78male 50′s roughened B once a day After 3 minutes, skin got moisturized skin and its texture was improved. Q79 female 20′s atopic  C once a dayAfter 3 minutes, erythema and scales  Itches were improved  dermatitiswere improved. immediately after  applilcation. Q80 female 50′sroughened B once a day After 3 minutes, skin roughness was Iches wee elminated  skin improved, skin got moisturized and its immediately after  texture was fimely improved. Skin gained application. resilence .After 7 days, large wrinkles and nasolabial fold because less deep. By continuing 4 weeks, spots faded and skin tone became brighter. Skin resilencewas improved. Large wrinkles became less  deep. Q81 male 30′s roughenedB once a day After 3 minutes, skin roughness and  skinredness were improved. Skin texture was finely improved. By continuing for 7 days, skin resilence was improved. SEQ ID NO 35: Q82 female 40′sroughened B once a day After 3 minutes, roughness skin and dry (CFGQKMDRISSSSGAGC) skin skin were promptly improved, skin texture was improved. Q83 male 60′s plaque  C twice a dayAfter 30 minutes, erythema, scales and  psoriasisinfiltration were improved. Q84 male 40′s plaque  C twice a dayAfter 20 minutes, erythema and scales  psoriasis were improved. Q85female 70′s plaque  C twice a day After5 minutes, erythema and scales psoriasis were remitted. 7 SEQ ID NO 36: Q86 female 10′s atopic  Conce a day After 2 minutes, linchenification was  After 2 minutes, (CFGQKMDRISSSSGLSC) dermatitis remarkably remitted, erythema, scales strong iches were  inflimation, cracks, scratch wound,  eliminated.etc. all remitted. After 5 minutes, improved with only mild infiltration  left. Q87 female 20′s atopic  Bonce a day After 1 minute, erythema, infiltration  After 2 minutes, dermatitis and papule remitted. After 4 minutes,  iches were infiltration was remitted with only  eliminated. mild erythema left. Q88male 10′s atopic  C once a day After 3 minutes, erythema, scales, After 3 minutes,  dermatitis infiltration and scratch wound were iches were  remitted. eliminated. Q89 female 40′s roughened B once a dayImmediately after application, itches  skinand redness were improved. After 3 minutes, skin got full and resilent,  and skin was improved. Q90 male50′s seborrhea C once a day, Immediately after application,seborrhea Itches were remitted dermatits 3 dayswas remitted. After 3 days, erythema  immediately after and seborrhea was remiited. application. SEQ ID NO 37: Q91 male 60′splaque  C once a day After 2 minutes, erythema and scales (CFGQKMDRISSSSGLAC) psoriasis were remitted. Q92 male 20′s atopic  Conce a day After 2 minutes, erythema and  Iches were eliminated dermatitis infiltration were remitted. Applied  immediately after prurigo twice. After 15 minutes, pringe nodularis  application.nodularis, on hip was remitted Q93 female 40′s roughened B once a dayAfter 3 minutes, roughened skin and dry  skinskin were improved promptly and skin  texture was improved. Q94 male40′s plaque  C once a day After 20 minutes, erythema and scales psoriasis were remitted. Q95 male 30′s atopic  C once a dayAfter 2 minutes, scratch wound  dermatitisepithelized erythema was remitted. SEQ ID NO 38: Q96 male 20′s atopic  Conce a day After 3 minutes, cracks was reduced in  immediately after (CFGQKMDRISSSSGLGC) dermatitis size. Erythema, scales and application, itches lichenification were remitted. and rawness were removed. Q97 female 40′s roughened B once a dayAfter 1 minute, dry skin was improved,  immediately after  skinskin got moiturized and full, and  application, itches skin texture was improved. were removed. Q98 female 200′s chronic Conce a day immediately after application, erythema  eczemaand scales were remitted. Q99 male 20′s atopic  C once a dayAfter 2 minutes, redness was improved.  After 1 minute, iches dermatitisAfter 3 minutes, erythema, infiltration  were eliminated. and papule were remitted. After 1  After 1 application,application, the effect lasted for 6  no itches for 6 days days with no recurrence. thereafter. Q100 female 50′s prurigo Conce a day After 3 minutes, nodes wereflatened,  Iches were eliminated nodularis, scratch wound became dry and epithelized  immediately after eczema with no more exudate. application. 8 SEQ ID NO 39: Q101 female50′s prurigo C once a day After 3 minutes, nodes were flattened.(CFSRKMDRISSSSGLGC) nodularis, eczema Q102 female 10′s chronic eczema Conce a day After 2 minutes, an intractable eczema that cannot be remitted by applying strongsteriod ointments was remitted, and erythema, scales and papule were reduced. Q103 female 50′s eczema Conce a day After 2 minutes, erythema, scales and pigmentation were remitted. Skin got full  and soft, with fresh color.Q104 female 20′s atopic  D once a dayScratch wound was epithelized Exudate was  Iches were eliminateddermatitis dried ad lichenification was remitted.  immediately after erythema and infiltration were remitted. application. Q105 male 20′satopic  B once a day After 3 minutes, erythema and infiltrationAfter 2 minutes,  dermatitis were remitted. With 1 application, the itches and tingling  effect persisted for 1 week with no pain were removed.   recurrence. The effect persistedfor 7 days with no  itches. SEQ ID NO 40: Q106 male 40′s atopic  Conce a day After 2 minutes, erythema, infiltration Iches were eliminated (CFTRKMDRISSSSGLGC) dermatitisand lichenification remitted. immediately after  application. Q107female 20′s roughened C 1 applicationAfter 1 minute, dry skin was improved,  lipscracks were less deep and apithelized.  Q108 female 40′s roughened Bonce a day After 3 minutes, redness and roughness  skin were improved.Q109 female 30′s healthy skin C once a dayAfter 3 minutes, dark-reddish skin tone was improved to turn fresh color. Dryness, tautnessm rawness and hot flash of skin were removed. With 1 applicationthe effect persisted for 1 week. 9 SEQ ID NO 41: Q110 male 10′s atopic C once a day immediately after application, erythema Iches were eliminated  (CFGRKMDRISSTSGLGC) dermatitis/and infiltration of eczema were remitted.  immediately after application. acne The effects were exhibited remarkably fast. After 3 minutes, erythema was further remitted. Acne was dried and  reduced in size. Q111 male 20′satopic  C once a day Immediately after application, erythema Iches were eliminated  dermatitis and infiltration were remitted. After immediately after  20 minutes, deep scratch wound was  application.epithelized. Q112 female 40′s roughened B 1 applicationAfter 1 minute, skintautness and rawness  lipswere improved and skin got moisturized. Q113 female 50′s atopic  Bonce a day Iches stopped in 30 seconds. Redness  dermatitisand edema were remitted in 1 minute. Q114 female 10′s roughened Bonce a day After 1 minute, redness was remitted. After 3 minutes, skinskin/acne After 3 minutes, skin roughness, zits,  tautness was removed.rough and soft skin texture were improved, skin got full end its texture was finelyimproved. Acne became dry and reduced in  size. Q115 female 40′sroughened B once a day Rawness vanished in 1 minute. Skin was  skinmoisturized. After 2 minutes, redness, roughness and stiffness were remarkably improved. By using for 1 weel, skin became brighter, flabbiness was improved. SEQ ID NO 42: Q116 female 40′sacne C once a day After 1 minute, redness was remitted.(CFGRKMDRISSASGLGC) Q117 male 10′s atopic  B once a dayAfter 3 minutes, erythema and  Iches were eliminated dermatitisinfiltration were remitted. immediately after  application. Q118 female50′s eczema C once a day After 3 minutes, erythema and dry skin remitted. SEQ ID NO 43: Q119 male 40′s atopic  C once a dayAfter 2 minutes, no discomfort, felt  After 3 minutes, (CFGRKMDRISSSTGLGC) dermatitis fresh. Erythema, infiltration and roughness and  lichenification were remitted. stiffness were removed, and itches  vanished. Q120 female 30′s roughened C once a day1 minute after application, dry skin  lipswas improved cracks became less deep  and epithelized. Q121 female 40′sroughened B once a day After 3 minutes, redness and dry skin  skinwere improved. Skin got moisturized promptly, and skin texture was improved. Skin rawness, stiffness and irritation were eliminated. Pores became less  distinguised. Q122 female 40′srosacae/acne C once a day After 7 minutes, acne was reduce and became dry. Q123 female 30′s eczema C once a dayAfter 1 minute, redness and infiltration  Iches were eliminated were remitted. immediately after  application. 10 SEQ ID NO 44: Q124female 60′s healthy skin B once a daySkin got moisturized promptly, wrinkles  (CFGRKMDRISSSAGLGC)became less deep, skin texture was improved.By using for 4 weeks, large wrinkles and nasolabial fold became less deep. Skin resilence and flabiness were improved. Spots faded. Skin tone became brighter. Q125 female 80′s eczema Conce a day After 1 minute, erythema and infiltration Iches were remitted  were remitted. immediately after  application.SEQ ID NO 45: Q126 female 40′s roughened B once a dayAfter 2 minutes, dryness, tautness and  (CFSRRMDRISSSSGLGC) skinrawness of skin were eliminated, skin got full, and wrinkles became less deep. Skin mained elasticity. By using for 5 days, flabbiness was improved. Skin became brighter, nasolabial fold became less deep. By using for 4 weeks, nasolabial fold became less deep. Skin resilence and flabbiness were improved. Spots faded and skin tone became brighter. SEQ ID NO 46: Q127 female 40′s eczema Conce a day After 2 minutes, erythema, scales and  (CFGRKMDRISSTSGLGC)infiltration were remitted. Skin got  softened. SEQ ID NO 47: Q128female 30′s chronic eczema C once a dayImmediately after application, erythema  (CFGRKMDRISSSTGLGC)and scales were remitted. SEQ ID NO 48: Q129 male 50′s eczema Conce a day After 2 minutes, erythema, scales and  (CFGRKMDRISSSSGIGC)infiltration were remitted. Skin got  moisturized. SEQ ID NO 49: Q130male 30′s chronic eczema C once a dayImmediately after application, erythema  (CFGRKMDRISSSSGLAC)and scales were remitted. SEQ ID NO 50: Q131 male 50′s eczema Conce a day After 2 minutes, erythema, scales and  (CFGRRMDRISSSTGLGC)infiltration were remitted. SEQ ID NO 51: Q132 female 60′s eczema Bonce a day After 3 minutes, erythema and  (CFGRRMDRISSSTGLGC)infiltration were remitted. SEQ ID NO 52: Q133 female 50′s eczema Conce a day After 2 minutes, erythema, scales and  (CFGRRMDRISSSSGIGC)infiltration were remitted. Skin got  moisturized. SEQ ID NO 53: Q134female 20′s eczema C once a dayAfter 1 minute, erythema and infiltration  (CFGRRMDRISSSSGLAC)were remitted. SEQ ID NO 54: Q135 female 30′s roughened B once a dayAfter 2 minutes, dryness, tautness and  (CFSRKMDRISSTSGIGC) skinrawness of skin were eliminated, skin got full and wrinkles became less deep.  Skin gained elasticity.SEQ ID NO 54: Q136 female 50′s eczema C once a dayAfter 2 minutes, erythema, scale and  (CFSRKMDRISSTSGIGC)infiltration were remitted. 11 SEQ ID NO 56: Q137 male 20′s roughened Bonce a day After 2 minutes, dryness, tautness and  (CFGRKMDRISSSTGIGC)skin rawness of skin were elimnated, skin got full, and wrinkles became less deep.  Skin gained elasticity.SEQ ID NO 57: Q138 female 40′s eczema C once a dayAfter 2 minutes, erythema, scales and  (CFGRKMDRISSSTGLAC)infiltration were remitted, scratch  wound was epithelized and dried.SEQ ID NO 58: Q139 female 30′s eczema C once a dayAfter 2 minutes, erythema, scales,  (CFGRKMDRISSSSGIAC)papule and infiltration were remitted. SEQ ID NO 59: Q140 male 60′sroughened B once a day After 2 minutes, dryness, tautness and (CFGRKMDRISSTSGLGC) skin rawness of skin were eliminated. Skin gained elasticity, wrinkles became less  deep. SEQ ID NO 60: Q141 female50′s chronic C once a day Immediately after application, erythema (CFGRRMDRISSSTGLGC) eczema and scales were remitted. SEQ ID NO 61: Q142female 30′s chronic C once a dayImmediately after application, erythema  (CFSRRMDRISSSSGIGC)and scales were remitted. SEQ ID NO 62: Q143 female 40′s roughened Bonce a day After 2 minutes, dryness, tautness and  (CFSRRMDRISSSSGLAC)skin rawness of skin were eliminated, skin got full, and wrinkles became less deep.  Skin gained elasticity.SEQ ID NO 63: Q144 male 50′s eczema B once a dayAfter 3 minutes, erythema and scales  (CFSRKMDRISSTSGIGC)infiltration were remitted, and skin got  softened. SEQ ID NO 64: Q145male 40′s chronic C once a day Immediately after application, erythema (CFSRKMDRISSTSGIGC) eczema and scales were remitted. SEQ ID NO 65: Q146female 40′s roughened C once a dayAfter 1 minute, skin got moisturized and  (CFGRKMDRISSSTGLAC) skinresilent, skin texture was improved. SEQ ID NO 66: Q147 male 50′s eczemaC once a day After 2 minutes, erythema, scales and  (CFGRKMDRISSSSGIAC)infiltration were remitted. SEQ ID NO 67: Q148 female 70′s roughened Bonce a day After 2 minutes, dryness, tautness and  (CFSRRMDRISSTSGIGC)skin rawness of skin were eliminated, skin got full, and wrinkles became less deep.  Skin gained elasticity.SEQ ID NO 68: Q149 female 30′s chronic C once a dayImmediately after application, erythema  (CFGRRMDRISSTSGLAC) eczemaand scales were remitted. SEQ ID NO 69: Q150 female 20′s eczema Conce a day After 2 minutes, erythema, scales,  (CFSRRMDRISSTSGIGC)infiltration were improved. 12 SEQ ID NO 70: 151 female 60′s chronic Conce a day immediately after application, erythema  (CFGRRMDRISSSTGLAC)eczema and scales were remitted. 152 female 30′s roughened C once a dayRedness vanished in 30 seconds. After 1  skinminute, skin texture improved finely.  SEQ ID NO 71:Pores became less distinguished. (CFSRRMDRISSTSGIGC) 153 male 50′sroughened A once a day After 2 minutes, dryness, tautness and  skinrawness of skin were eliminated, skin got full and wrinkles became less deep.  Skin gained elasticity.SEQ ID NO 72: 154 male 50′s chronic C once a dayImmediately after application, erythema,  (CFSRRMDRISSTSGLAC) eczemascales and papule were remitted. SEQ ID NO 73: 155 male 20′s chronic Conce a day Immdediately after application, erythema  (CFSRRMDRISSSTGIGC)eczema and scales were remitted. SEQ ID NO 74: 156 female 30′s roughenedC once a day After 1 minute, rawness and hot flash  (CFGRRMDRISSSTGLAC)skin were eliminated, skin got moisturized and its texture improved, wrinkles faded  and skin gained elasticity.157 male 60′s chronic D once a dayIches were eliminated immediately after  eczemaapplication. Redness was remitted. SEQ ID NO 75: 158 female 60′s eczemaC once a day After 2 minutes, erythema, scalesm  (CFGRRMDRISSSSGIAC)papule, infiltration were remitted. SEQ ID NO 43: 258 male 40′s atopic C once a day After 2 minutes, cracks were epithelized (CFGRKMDRISSSTGLGC) dermatitis with no more pains and itches. Erythema and and scales were also remitted. SEQ ID NO 19: (CFGRRMDRISSSSLGC)SEQ ID NO 43: 358 male 40′s eczema once a dayAfter 2 minutes, wound was epithelized  (CFGRKMDRISSSTGLGC)quickly, and erythema, scales and  andinfiltration were remarkably remitted. SEQ ID NO 99: In SEQ ID NOS16-75, 1st Cys and 17th Cys from left form a disulfide bond.

The result described above demonstrated that the cyclic peptide of theinvention exhibits a similar effect as the cyclic peptide expressed byFormula I-a even if some amino acids of the cyclic peptide have beenreplaced. Moreover, a similar effect could be obtained by applying thereplaced cyclic peptide in a mixture as long as an effective amount ofthe cyclic peptide is applied.

Specifically, in subjects having atopic dermatitis or eczema, effectswere observed immediately after applying 3 μg/ml to 500 μg/ml toaffected site, including elimination of itches, epithelialization atinfiltrated site, papule or scratch, termination of exudation,epithelialization of a crack. Moreover, a single application induced apersistent effect for a week or more.

When it was applied to healthy or roughened skin, it exerted amoisturizing effect immediately or within a few minutes afterapplication, improving dryness, providing and keeping skin and mucosawith moderate elasticity and flexibility, softening skin, giving skin ormucosa resilience and firmness. Also, fine wrinkles and flabbiness areimproved at the applied site, and dullness and spots are further faded.A single application induced a persistent effect for a week or more.

9.3 Confirming Nasal Effect

The obtained cyclic peptide was prepared in physiological saline at 3,15, 30, 100 or 500 μg/ml, and 0.1 ml each was applied to nasal cavity.Each of formulations A to E in the table indicates the concentration ofthe cyclic peptide at 3, 15, 30, 100 or 500 μg/ml, respectively. Theabsence of the effect upon the application of physiological salinewithout the peptide confirmed the effect was not a placebo effect.

TABLE 13-1 SEQ ID NO (amino acid sequence) of  Diagnostic Formu-Diagnostic impression on Itches cyclic peptides ID Sex Age impressionlation Treatment skin after application after application SEQ ID NO 16:P1 female 10's allergic C once a day, 0.1 mlFrom immediately after nasal Itches were remitted (CFVRKMDRISSS rhinitisapplied to each application, and nose got immediately after nasal SGLGC)nasal cavity cleared, rhinorrhea stopped. application, and eliminatedThere was no irritationg after 1 minute. symptoms, and the effectpersisted for all day by 1 nasal application. SEQ ID NO 17: P2 male 30'sallergic D once a day, 0.1 ml From immediately after nasalItches were remitted (CFGQKMDRISSS rhinitis applied to eachapplication, and nose got immediately after nasal SGLGC) nasal cavitycleared, rhinorrhea stopped. application, and eliminatedThere was no irritationg after 1 minute. symptoms, and the effectpersisted for all day by 1 nasal application. SEQ ID NO 18: P3 male 50'sallergic C once a day, 0.1 ml After 3 minutes, and nose (CFGHKMDRISSSrhinitis applied to each got cleared, rhinorrhea SGLGC) nasal cavitystopped. There was no irritationg symptoms, andthe effect persisted for all day by 1 nasal application. SEQ ID NO 19:P4 female 40's chronic B once a day, 0.1 ml After 1 minute, and nose got(CFGRRMDRISSS rhinitis, applied to each cleared, nasal discharge SGLGC)perennial nasal cavity stopped. There was no rhinitisirritating symptoms. P5 female 70's allergic B once a day, 0.1 mlAfter 2 minutes, and nose Itches were remitted rhinitis applied to eachgot cleared, and no nasal immediately after nasal nasal cavitydischarge came out by application. blowing nose. SEQ ID NO 20: P6 female40's allergic B once a day, 0.1 ml After 1 minute, nasal (CFGRKLDRISSSrhinitis applied to each obstruction was improved, SGLGC) nasal cavityand nose got cleared. The effect persisted for all day. SEQ ID NO 21: P7female 20's allergic C once a day, 0.1 ml After 1 minute, and nose got(CFGRKIDRISSS rhinitis applied to each cleared. Nasal discharge SGLGC)nasal cavity stopped. SEQ ID NO 22: P8 female 50's allergic Eonce a day, 0.1 ml Immediately after nasal Immediately after nasal(CFGRKMDRVSSS rhinitis applied to each application, and nose gotapplication, restless itches SGLGC) nasal cavity cleared.and sneezing stopped. P9 female 40's chronic C once a day, 0.1 mlAfter 20 seconds, and nose rhinitis, applied to eachgot cleared. No nasal perennial nasal cavity discharge after 1 minute.rhinitis There was no irritating symptoms. SEQ ID NO 23: P10 female 30'sallergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRIGSSrhinitis applied to each cleared. Nasal discharge SGLGC) nasal cavitystopped. SEQ ID NO 24: P11 female 20's allergic C once a day, 0.1 mlAfter 1 minute, and nose got (CFGRKMDRISAS rhinitis applied to eachcleared. Nasal discharge SGLGC) nasal cavity stopped. SEQ ID NO 25: P12female 20's allergic A once a day, 0.1 ml After 2.5 minutes, and nose(CFGRKMDRISSQ rhinitis applied to each got cleared. SGLGC) nasal cavitySEQ ID NO 26: P13 female 50's allergic C once a day, 0.1 mlAfter 1 minute, nose got (CFGRKMDRISSV rhinitis applied to eachcleared and refreshed. After SGLGC) nasal cavity 2 minutes, no nasaldischarge by blowing. SEQ ID NO 27: P14 male 20's allergic Conce a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRISSL rhinitisapplied to each cleared. SGLGC) nasal SEQ ID NO 28: P15 female 40'sallergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRISSIrhinitis applied to each cleared. Nasal discharge SGLGC) nasal cavitystopped. SEQ ID NO 29: P16 male 50's allergic C once a day, 0.1 mlAfter 1 minute, and nose got (CFGRKMDRISSM rhinitis applied to eachcleared. Nasal discharge SGLGC) nasal cavity stopped. SEQ ID NO 30: P17female 30's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea(CFGRKMDRISSS rhinitis applied to each stopped. VGLGC) nasal cavitySEQ ID NO 31: P18 female 10's allergic D once a day, 0.1 mlAfter 30 seconds, rhinorrhea (CFGRKMDRISSS rhinitis applied to eachstopped. After 1 minute, no SRLGC) nasal cavitynasal discharge by blowing. SEQ ID NO 32: P19 male 40's allergic Conce a day, 0.1 ml After 1 minute, nasal (CFGRKMDRISSS rhinitisapplied to each obstruction was improved, SGMGC) nasal cavityand nose got cleared. The effect persisted for all day. P20 female 40'schronic C once a day, 0.1 ml Immediately after nasal rhinitis,applied to each application nose got perennial nasal cavitycleared. The effect was rhinitis remarkable and persistedfor 7 days after 1 nasal application. SEQ ID NO 33: P21 female 40'sallergic C once a day, 0.1 ml After 30 seconds, rhinorrheaSneezing and itches in deep (CFGRKMDRISSS rhinitis applied to eachstopped. After 1 minute, no inside nose stopped. SGIGC) nasal cavitynasal discharge by blowing. SEQ ID NO 34: P22 female 40's allergic Conce a day, 0.1 ml After 2.5 minutes, and nose No irritating symptoms.(CFGRKMDRISSS rhinitis applied to each got cleared. SGVGC) nasalSEQ ID NO 35: P23 female 30's allergic C once a day, 0.1 mlAfter 1 minute, nasal (GFGRKMDRISSS rhinitis applied to eachobstruction was improved, SGAGC) nasal cavity and nose got cleared. Theeffect persisted for all day. P24 female 40's chronic Donce a day, 0.1 ml Immediately after nasal rhinitis, applied to eachapplication, nose got perennial nasal cavity refreshed and cleared.rhinitis SEQ ID NO 36: P25 female 40's allergic C once a day, 0.1 mlImmediately after nasal Immediately after nasal (CFGRKMDRISSS rhinitisapplied to each application, and nose got application, itches wereSGLSC) nasal cavity cleared. removed. No irritating symptoms.SEQ ID NO 37: P26 female 40's allergic C once a day, 0.1 mlAfter 30 seconds, rhinorrhea Immediate effect to remove (CFGRKMDRISSSrhinitis applied to each stopped. Nose got cleared itches. SGLAC)nasal cavity and felt good. Other nose drops causes this subject tosneeze due to some irritant, but the inventive nose drophad no irritation. The effect persisted for 1 weekby 1 nasal application. P27 female 40's chronic C once a day, 0.1 mlNose got cleared and rhinitis, applied to each rhinorrhea stopped in 30perennial nasal cavity seconds. rhinitis SEQ ID NO 38: P28 male 30'schronic C once a day, 0.1 ml After 2 minutes, rhinorrhea (CFARKMDRISSSrhinitis applied to each stopped. Nasal obstruction SGLGC) nasal cavitywas improved to allow nasal breathing. SEQ ID NO 39: P29 male 40'sallergic C once a day, 0.1 ml Immediately after nasal (CFSRKMDRISSSrhinitis applied to each application, nose got SGLGC) nasal cavityrefreshed and cleared. No nasal discharge after 30 seconds.SEQ ID NO 40: P30 female 30's allergic C once a day, 0.1 mlAfter 2.5 minutes, and nose (CFTRKMDRISSS rhinitis applied to eachgot cleared. SGLGC) nasal SEQ ID NO 41: P31 female 20's allergic Conce a day, 0.1 ml After 1 minute, rhinorrhea (CFGRKMDRISST rhinitisapplied to each stopped. There was no SGLGC) nasal cavityirritating symptoms. SEQ ID NO 42: P32 female 10's allergic Conce a day, 0.1 ml Immediately after nasal (CFGRKMDRISSA rhinitisapplied to each application, and nose got SGLGC) nasal cavity cleared.SEQ ID NO 43: P33 male 20's allergic C once a day, 0.1 mlImmediately after nasal (CFGRKMDRISSS rhinitis applied to eachapplication, and nose got TGLGC) nasal cavitycleared. After 1 minute, no nasal discharge even by twisting nose.SEQ ID NO 44: P34 male 40's allergic C once a day, 0.1 mlAfter 1 minute, rhinorrhea (CFGRKMDRISSS rhinitis applied to eachstopped. There was no AGLGC) nasal cavity irritating symptoms.SEQ ID NO 45: P35 male 60's allergic C once a day, 0.1 mlAfter 1 minute, and nose got (CFSRRMDRISSS rhinitis applied to eachcleared. SGLGC) nasal SEQ ID NO 47: P36 male 50's allergic Conce a day, 0.1 ml After 1 minute, rhinorrhea (CFSRKMDRISSS rhinitisapplied to each stopped. There was no TGLGC) nasal cavityirritating symptoms. SEQ ID NO 48: P37 female 50's allergic Conce a day, 0.1 ml After 1 minute, and nose got (CFSRKMDRISSS rhinitisapplied to each cleared. Nasal discharge SGIGC) nasal cavity stopped.SEQ ID NO 49: P38 female 40's allergic C once a day, 0.1 mlImmediately after nasal (CFSRKMDRISSS rhinitis applied to eachapplication, and nose got SGLAC) nasal cavity cleared. SEQ ID NO 50: P39female 10's allergic C once a day, 0.1 ml Immediately after nasal(CFGRRMDRISST rhinitis applied to each application, and nose got SGLGC)nasal cavity cleared. SEQ ID NO 51: P40 male 20's chronic Conce a day, 0.1 ml After 1.5 minutes, nasal (CFGRRMDRISSS rhinitisapplied to each obstruction was improved, TGLGC) nasal cavityand nose got cleared. SEQ ID NO 52: P41 male 30's allergic Conce a day, 0.1 ml After 1 minute, rhinorrhea (CFGRRMDRISSS rhinitisapplied to each stopped. There was no SGIGC) nasal cavityirritating symptoms. SEQ ID NO 53: P42 male 40's allergic Conce a day, 0.1 ml Immediately after nasal (CFGRRMDRISSS rhinitisapplied to each application, and nose got SGLAC) nasal cavity cleared.SEQ ID NO 54: P43 female 30's allergic C once a day, 0.1 mlImmediately after nasal (CFGRKMDRISST rhinitis applied to eachapplication, and nose got SGIGC) nasal cavity cleared. SEQ ID NO 55: P44male 50's allergic C once a day, 0.1 ml After 1 minute, and nose got(CFGRKMDRISST rhinitis applied to each cleared. Nasal discharge SGLAC)nasal cavity stopped. SEQ ID NO 56: P45 male 30's allergic Conce a day, 0.1 ml Immediately after nasal (CFGRKMDRISSS rhinitisapplied to each application, and nose got TGIGC) nasal cavity cleared.SEQ ID NO 57: P46 female 40's allergic C once a day, 0.1 mlAfter 1 minute, and nose got (CFGRKMDRISSS rhinitis applied to eachcleared. Nasal discharge TGLAC) nasal cavity stopped. SEQ ID NO 58: P47female 40's allergic C once a day, 0.1 ml After 30 seconds, rhinorrheaItches were removed in (CFGRKMDRISSS rhinitis applied to eachstopped, with no more 30 seconds. SGIAC) nasal cavitysneezing. There was no irritating symptoms. SEQ ID NO 59: P48 female20's allergic C once a day, 0.1 ml Immediately after nasal (CFSRRMDRISSTrhinitis applied to each application, and nose got SGLGC) nasal cavitycleared. SEQ ID NO 60: P49 female 40's allergic C once a day, 0.1 mlAfter 1 minute, and nose got (CFSRRMDRISSS rhinitis applied to eachcleared. Nasal discharge TGLGC) nasal cavity stopped. SEQ ID NO 61: P50male 30's allergic C once a day, 0.1 ml Immediately after nasal(CFSRRMDRISSS rhinitis applied to each application, and nose got SGIGC)nasal cavity cleared. SEQ ID NO 62: P51 female 40's allergic Conce a day, 0.1 ml After 1 minute, rhinorrhea (CFSRRMDRISSS rhinitisapplied to each stopped, with no more SGLAC) nasal cavitysneezing. There was no irritating symptoms. SEQ ID NO 63: P52 female30's allergic C once a day, 0.1 ml Immediately after nasal (CFSRKMDRISSTrhinitis applied to each application, and nose got SGIGC) nasal cavitycleared. SEQ ID NO 64: P53 male 60's allergic C once a day, 0.1 mlAfter 1 minute, rhinorrhea (CFSRKMDRISSS rhinitis applied to eachstopped, with no more TGIGC) nasal cavity sneezing. There was noirritating symptoms. SEQ ID NO 65: P54 female 30's allergic Conce a day, 0.1 ml After 1 minute, and nose got (CFSRKMDRISSS rhinitisapplied to each cleared. Nasal discharge TGLAC) nasal cavity stopped.SEQ ID NO 66: P55 female 30's allergic C once a day, 0.1 mlImmediately after nasal (CFSRKMDRISSS rhinitis applied to eachapplication, and nose got SGIAC) nasal cavity cleared. SEQ ID NO 67: P56female 40's allergic C once a day, 0.1 ml After 1 minute, and nose(CFGRRMDRISST rhinitis applied to each got cleared. Nasal SGIGC)nasal cavity discharge stopped. SEQ ID NO 68: P57 male 60's allergic Conce a day, 0.1 ml After 1 minute, rhinorrhea (CFGRRMDRISST rhinitisapplied to each stopped, with no more SGLAC) nasal cavitysneezing. There was no irritating symptoms. SEQ ID NO 69: P58 male 20'sallergic C once a day, 0.1 ml Immediately after nasal (CFGRRMDRISSSrhinitis applied to each application, and nose got TGIGC) nasal cavitycleared. SEQ ID NO 70: P59 female 40's allergic C once a day, 0.1 mlImmediately after nasal (CFGRRMDRISSS rhinitis applied to eachapplication, and nose got TGLAC) nasal cavity cleared. SEQ ID NO 71: P60female 30's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea(CFSRRMDRISST rhinitis applied to each stopped, with no more SGIGC)nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 72:P61 female 40's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea(CFSRRMDRISST rhinitis applied to each stopped, with no more SGLAC)nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 73:P62 female 20's allergic C once a day, 0.1 ml Immediately after nasal(CFSRRMDRISSS rhinitis applied to each application, and nose got TGIGC)nasal cavity cleared. SEQ ID NO 74: P63 female 40's allergic Conce a day, 0.1 ml After 1 minute, rhinorrhea (CFSRRMDRISSS rhinitisapplied to each stopped, with no more TGLAC) nasal cavitysneezing. There was no irritating symptoms. SEQ ID NO 75: P64 male 20'sallergic C once a day, 0.1 ml After 1 minute, and nose (CFSRRMDRISSSrhinitis applied to each got cleared. Nasal SGIAC) nasal cavitydischarge stopped. In SEQ ID NOS 16-75, 1st Cys and 17th Cys from leftform a disulfide bond.

The result described above demonstrated that the cyclic peptide of theinvention exhibits a similar effect as the cyclic peptide expressed byFormula I-a even if some amino acids of the cyclic peptide have beenreplaced.

Specifically, when it was applied to nasal cavity mucosa and/orparanasal cavity mucosa, it can improve or prevent various symptomsassociated with rhinitis such as nasal obstruction, rhinorrhea, sneezingand pruritus. Rhinitis also includes, without being particularlylimited, for example, infectious rhinitis including acute rhinitis andchronic rhinitis; hypersensitive non-infectious rhinitis includingcombined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea,congestive rhinitis, edematous rhinitis and dry-nose type rhinitis;irritant-induced rhinitis including physical rhinitis, chemical rhinitisand radiation rhinitis; and atrophic rhinitis and idiopathicgranulomatous rhinitis; sinusitis such as acute sinusitis, chronicsinusitis (maxillary empyema), eosinophilic sinusitis and paranasalcavity mycosis. The external preparation can be used for the purpose oftreating or preventing one ore more of the above.

Combined rhinitis includes, for example, allergic rhinitis includingperennial allergic rhinitis and seasonal allergic rhinitis, andnonallergic rhinitis including vasomotor (essential) rhinitis andeosinophilic rhinitis. Rhinitis with rhinorrhea includes, for example,gustatory rhinitis, cold air-induced rhinitis and senile rhinitis.

Congestive rhinitis includes, for example, drug-induced rhinitis,psychogenic rhinitis, gestational rhinitis, endocrine rhinitis and coldrhinitis.

Edematous rhinitis includes, for example, aspirin-hypersensitiverhinitis.

Among those mentioned above, it exerts an excellent effect specificallyon hypersensitive non-infectious rhinitis, irritant-induced rhinitis andsinusitis, preferably on hypersensitive non-infectious rhinitis andchronic sinusitis, more preferably on combined (flower hypersensitivity)rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematousrhinitis and dry-nose type rhinitis, and chronic sinusitis. Therefore,the external preparation of the present invention can be used for thepurpose of treating or preventing at least one or more rhinitis selectedfrom the above-mentioned group.

In terms of its symptoms, for its effects as described above, it can beused for any of rhinitis with sneezing/rhinorrhea, rhinitis with nasalobstruction and rhinitis with both symptoms.

Immediately after a single application of 0.1 ml of 3 μg/ml to 500 μg/mlpreparation was applied to the affected site in each nasal cavity, nasaldischarge was stopped, nasal obstruction was improved, itches in nasalcavity mucosa or sneezing were stopped (immediate effect). A singleapplication of a nasal drop induced persistent effects lasting for oneday, or for a week or more in some cases (long-lasting effect). Inaddition, because it causes no local irritation at all, it can be usedsafely even in a subject who is not desired to use a nasal dropcomprising rhinitis therapeutic that has conventionally been used suchas steroid drug due to its strong local irritation.

Examples of multiple replaceable amino acids in the cyclic peptide ofthe invention are summarized below, based on working examples asdescribed above, without not being limited thereto.

TABLE 32 Candidate multiple replacements of B ring Number Example of ofResi- Resi- Resi- Resi- replaced SEQ replace- due Amino due Amino dueAmino due Amino amino acid ID ment ID acid ID acid ID acid ID acidsequence NO: 2 3 A, S 5 R CFSRRMDRISSSSGLGC 45 3 A, S 12 Q, TCFSRKMDRISSTSGLGC 46 3 A, S 13 T CFSRKMDRISSSTGLGC 47 3 A, S 15 M, I, CFSRKMDRISSSSGIGC 48 V, A 3 A, S 16 A, S CFSRKMDRISSSSGLAC 49 5 R 12Q, T CFGRRMDRISSTSGLGC 50 5 R 13 T CFGRRMDRISSSTGLGC 51 5 R 15 M, I,CFGRRMDRISSSSGIGC 52 V, A 5 R 16 A, S CFCRRMDRISSSSGLAC 53 12 Q, T 15M, I, CFGRKMDRISSTSGIGC 54 V, A 12 Q, T 16 A, S CFGRKMDRISSTSGLAC 55 13T 15 M, I, CFGRKMDRISSSTGIGC 56 V, A 13 T 16 A, S CFGRKMDRISSSTGLAC 5715 M, I,  16 A, S CFGRKMDRISSSSGIAC 58 V, A 3 3 A, S 5 R 12 Q, TCFSRRMDRISSTSGLGC 59 3 A, S 5 R 13 T CFSRRMDRISSSTGLGC 60 3 A, S 5 R 15M, I, V, A CFSRRMDRISSSSGIGC 61 3 A, S 5 R 16 A, S CFSRRMDRISSSSGLAC 623 A, S 12 Q, T 15 M, I, V, A CFSRKMDRISSTSG1GC 63 3 A, S 13 T 15M, I, V, A CFSRKMDRISSSTGIGC 64 3 A, S 13 T 16 A, S CFSRKMDRISSSTGLAC 653 A, S 15 M, I, 16 A, S CFSRKMDRISSSSGLAC 66 V, A 5 R 12 Q, T 15M, I, V, A CFGRRMDRISSTSGIGC 67 5 R 12 Q, T 16 A, S CFGRRMDRISSTSGIAC 685 R 13 T 15 M, I, V, A CFGRRMDRISSSTGIGC 69 5 R 13 T 16 A, SCFGRRMDRISSSTGLAC 70 5 R 15 M, I, 16 A, S CFGRRMDRISSSSGLAC 71 V, A 4 3A, S 5 R 12 Q, T 15 M, I,  CFSRRMDR1SSTSGIGC 72 V, A 3 A, S 5 R 12 Q, T16 A, S CFSRRMDRISSTSGLAC 73 3 A, S 5 R 13 T 15 M, I,  CFSRRMDRISSSTGIGC74 V, A 3 A, S 5 R 13 T 16 A, S CFSRRMDRISSSTGLAC 75 3 A, S 5 R 15M, I, V, A 16 A, S CFSRRMDRISSSSGIAC 76

1.-15. (canceled)
 16. A method of using an external preparationcomprising applying the external preparation to skin and/or mucosa of asubject, wherein the external preparation comprises a cyclic peptidehaving an amino acid sequence expressed by the Formula I:

wherein, X¹ denotes Gly, Val, Ala, Ser or Thr, X² denotes Arg, Gln orHis, X³ denotes Lys or Arg, X⁴ denotes Met, Leu or Ile, X⁵ denotes Ileor Val, X⁶ denotes Ser or Gly, X⁷ denotes Ser or Ala, X⁸ denotes Ser,Gln, Val, Ala, Thr, Leu, Ile or Met, X⁹ denotes Ser, Val, Ala or Thr,X¹⁰ denotes Gly or Arg, X¹¹ denotes Leu, Met, Ile, Val or Ala, X¹²denotes Gly, Ser or Ala, and the line connecting two Cys denotes adisulfide bond, and wherein said amino acid sequence does not have apeptide bond that is not between the amino acids constituting said aminoacid sequence, or a derivative thereof or a pharmaceutically acceptablesalt thereof.
 17. The method of using the external preparation accordingto claim 16, wherein the mucosa is labial, oral, nasal, ocular orvaginal mucosa.
 18. The method of using the external preparationaccording to claim 16 for treating and/or preventing dermatitis, foralleviating or resolving itch, for treating eczematous or other erosionor ulcer, or for skin-care.
 19. The method of using the externalpreparation according to claim 16 for treating and/or preventingalopecia, and/or for stimulating hair growth, and/or for growing hair.20. The method of using the external preparation according to claim 16for treating and/or preventing rhinitis.
 21. (canceled)
 22. (canceled)23. The method according to claim 16, wherein X¹-X¹² are selected fromthe group consisting of following (1)-(12): (1) X¹ denotes Gly, (2) X²denotes Arg, Gln or His, (3) X³ denotes Lys or Arg, (4) X⁴ denotes Met,Leu or Ile, (5) X⁵ denotes Ile, (6) X⁶ denotes Ser or Gly, (7) X⁷denotes Ser or Ala, (8) X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile orMet, (9) X⁹ denotes Ser, Val, Ala or Thr, (10) X¹⁰ denotes Gly or Arg,(11) X¹¹ denotes Leu, and (12) X¹² denotes Gly, or a derivative thereofor a pharmaceutically acceptable salt thereof.
 24. The method accordingto claim 16 selected from the cyclic peptides of SEQ ID NOs: 3-8 and SEQID NOs: 16-75, or a derivative thereof or a pharmaceutically acceptablesalt thereof.
 25. The method according to claim 16, wherein thederivative is substituted by a substituent which is capable of replacinga hydrogen atom, hydroxyl group, carboxy group, amino group or iminogroup in the cyclic peptide.
 26. The method according to claim 16,wherein the cyclic peptide is formed by deleting 1 to 4 amino acids inthe cyclic peptide expressed by any one of the Formulae (I-a)-(I-e), orby replacing them with or adding them other amino acids, and wherein thecyclic peptide has an equal function with the cyclic peptide expressedby each of said formulae.
 27. The method according to claim 16, whereinthe external preparation is an ingredient for a dermatitis therapeutic,dermatitis prophylactic, antipruritic, antiphlogistic, woundepithelialization-accelerating agent or skin-care product.
 28. Themethod according to claim 27, wherein the skin-care product is used formoisturizing, and/or for preventing or improving rough skin, and/or forsebum/acne care, and/or for alleviating irritation/anti-inflammation,and/or for skin-lightening, and/or for anti-aging, and/or forpreventing/alleviating ultraviolet lesion, and/or for slimming, and/orfor skin-cleansing.
 29. The method according to claim 16, wherein theexternal preparation is a bath agent, a body-cleansing agent or ahair-cleansing agent.
 30. The method according to claim 16, wherein theexternal preparation is an alopecia therapeutic, an alopeciaprophylactic, a hair growing agent and/or a hair growth stimulant. 31.The method according to claim 16, wherein the external preparation is arhinitis therapeutic and/or rhinitis prophylactic.
 32. The methodaccording to claim 16, wherein the external preparation is a cosmetic.33. The method according to claim 16, wherein the external preparationis a solid, semi-solid, powder, liquid, spray, ointment, cream,emulsion, gel or patch formulation.
 34. The method according to claim16, wherein the external preparation is used as a pharmaceuticalproduct, a quasi-drug or a cosmetic product.